β1,3-galactosyltransferases from C. jejuni

ABSTRACT

This invention provides prokaryotic glycosyltransferases, including a bifunctional sialyltransferase that has both an α2,3- and an α2,8-activity. A β1,4-GalNAc transferase and a β1,3-galactosyltransferase are also provided by the invention, as are other glycosyltransferases and enzymes involved in synthesis of lipooligosaccharide (LOS). The glycosyltransferases can be obtained from, for example,  Campylobacter  species, including  C. jejuni.  In additional embodiments, the invention provides nucleic acids that encode the glycosyltransferases, as well as expression vectors and host cells for expressing the glycosyltransferases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application divisional application of U.S. patent application Ser.No. 10/303,134, filed Nov. 21, 2002, now U.S. Pat. No. 6,825,019; whichis a divisional application of U.S. patent application Ser. No.09/816,028, filed Mar. 21, 2001, now U.S. Pat. No. 6,699,705; which is acontinuation-in-part of U.S. application Ser. No. 09/495,406, filed Jan.31, 2000, now U.S. Pat. No. 6,503,744; which claims benefit of U.S.Provisional Application No. 60/118,213, which was filed on Feb. 1, 1999;all four applications are incorporated herein by reference for allpurposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of enzymatic synthesis ofoligosaccharides, including gangliosides and ganglioside mimics.

2. Background

Gangliosides are a class of glycolipids, often found in cell membranes,that consist of three elements. One or more sialic acid residues areattached to an oligosaccharide or carbohydrate core moiety, which inturn is attached to a hydrophobic lipid (ceramide) structure whichgenerally is embedded in the cell membrane. The ceramide moiety includesa long chain base (LCB) portion and a fatty acid (FA) portion.Gangliosides, as well as other glycolipids and their structures ingeneral, are discussed in, for example, Lehninger, Biochemistry (WorthPublishers, 1981) pp. 287–295 and Devlin, Textbook of Biochemistry(Wiley-Liss, 1992). Gangliosides are classified according to the numberof monosaccharides in the carbohydrate moiety, as well as the number andlocation of sialic acid groups present in the carbohydrate moiety.Monosialogangliosides are given the designation “GM”,disialogangliosides are designated “GD”, trisialogangliosides “GT”, andtetrasialogangliosides are designated “GQ”. Gangliosides can beclassified further depending on the position or positions of the sialicacid residue or residues bound. Further classification is based on thenumber of saccharides present in the oligosaccharide core, with thesubscript “1” designating a ganglioside that has four saccharideresidues (Gal-GalNAc-Gal-Glc-Ceramide), disaccharide (Gal-Glc-Ceramide)and monosaccharide (Gal-Ceramide) gangliosides, respectively.

Gangliosides are most abundant in the brain, particularly in nerveendings. They are believed to be present at receptor sites forneurotransmitters, including acetylcholine, and can also act as specificreceptors for other biological macromolecules, including interferon,hormones, viruses, bacterial toxins, and the like. Gangliosides are havebeen used for treatment of nervous system disorders, including cerebralischemic strokes. See, e.g., Mahadnik et al. (1988) Drug DevelopmentRes. 15: 337–360; U.S. Pat. Nos. 4,710,490 and 4,347,244; Horowitz(1988) Adv. Exp. Med. and Biol. 174: 593–600; Karpiatz et al. (1984) AdvExp. Med. and Biol. 174: 489–497. Certain gangliosides are found on thesurface of human hematopoietic cells (Hildebrand et al. (1972) Biochim.Biophys. Acta 260: 272–278; Macher et al. (1981) J. Biol. Chem. 256:1968–1974; Dacremont et al. Biochim. Biophys. Acta 424: 315–322; Klocket al. (1981) Blood Cells 7: 247) which may play a role in the terminalgranulocytic differentiation of these cells. Nojiri et al. (1988) J.Biol. Chem. 263: 7443–7446. These gangliosides, referred to as the“neolacto” series, have neutral core oligosaccharide structures havingthe formula [Galβ-(1,4)GlcNAcβ(1,3)]_(n)Galβ(1,4)Glc, where n=1–4.Included among these neolacto series gangliosides are 3′-nLM₁(NeuAcα(2,3)Galβ(1,4)GlcNAcβ(1,3)Galβ(1,4)-Glcβ(1,1)-Ceramide) and6′-nLM₁ (NeuAcα(2,6)Galβ(1,4)GlcNAcβ(1,3)Galβ(1,4)-Glcβ(1,1)-Ceramide).

Ganglioside “mimics” are associated with some pathogenic organisms. Forexample, the core oligosaccharides of low-molecular-weight LPS ofCampylobacter jejuni O:19 strains were shown to exhibit molecularmimicry of gangliosides. Since the late 1970s, Campylobacter jejuni hasbeen recognized as an important cause of acute gastroenteritis in humans(Skirrow (1977) Brit. Med. J. 2: 9–11). Epidemiological studies haveshown that Campylobacter infections are more common in developedcountries than Salmonella infections and they are also an importantcause of diarrheal diseases in developing countries (Nachamkin et al.(1992) Campylobacter jejuni: Current Status and Future Trends. AmericanSociety for Microbiology, Washington, D.C.). In addition to causingacute gastroenteritis, C. jejuni infection has been implicated as afrequent antecedent to the development of Guillain-Barré syndrome, aform of neuropathy that is the most common cause of generalyzedparalysis (Ropper (1992) N. Engl. J. Med. 326: 1130–1136). The mostcommon C. jejuni serotype associated with Guillain-Barré syndrome isO:19 (Kuroki (1993) Ann. Neurol. 33: 243–247) and this prompted detailedstudy of the lipopolysaccharide (LPS) structure of strains belonging tothis serotype (Aspinall et al. (1994a) Infect. Immun. 62: 2122–2125;Aspinall et al. (1994b) Biochemistry 33: 241–249; and Aspinall et al.(1994c) Biochemistry 33: 250–255).

Terminal oligosaccharide moieties identical to those of GD1a, GD3, GM1and GT1a gangliosides have been found in various C. jejuni O:19 strains.C. jejuni OH4384 belongs to serotype O:19 and was isolated from apatient who developed the Guillain-Barré syndrome following a bout ofdiarrhea (Aspinall et al. (1994a), supra.). It was showed to possess anouter core LPS that mimics the tri-sialylated ganglioside GT1a.Molecular mimicry of host structures by the saccharide portion of LPS isconsidered to be a virulence factor of various mucosal pathogens whichwould use this strategy to evade the immune response (Moran et al.(1996a) FEMS Immunol. Med. Microbiol. 16: 105–115; Moran et al. (1996b)J. Endotoxin Res. 3: 521–531).

Consequently, the identification of the genes involved in LPS synthesisand the study of their regulation is of considerable interest for abetter understanding of the pathogenesis mechanisms used by thesebacteria. Moreover, the use of gangliosides as therapeutic reagents, aswell as the study of ganglioside function, would be facilitated byconvenient and efficient methods of synthesizing desired gangliosidesand ganglioside mimics. A combined enzymatic and chemical approach tosynthesis of 3′-nLM₁ and 6′-nLM₁ has been described (Gaudino and Paulson(1994) J. Am. Chem. Soc. 116: 1149–1150). However, previously availableenzymatic methods for ganglioside synthesis suffer from difficulties inefficiently producing enzymes in sufficient quantities, at asufficiently low cost, for practical large-scale ganglioside synthesis.Thus, a need exists for new enzymes involved in ganglioside synthesisthat are amenable to large-scale production. A need also exists for moreefficient methods for synthesizing gangliosides. The present inventionfulfills these and other needs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1C show lipooligosaccharide (LOS) outer core structures from C.jejuni O:19 strains. These structures were described by Aspinall et al.(1994) Biochemistry 33, 241–249, and the portions showing similaritywith the oligosaccharide portion of gangliosides are delimited by boxes.FIG. 1A: LOS of C. jejuni O:19 serostrain (ATCC #43446) has structuralsimilarity to the oligosaccharide portion of ganglioside GD1a. FIG. 1B:LOS of C. jejuni O:19 strain OH4384 has structural similarity to theoligosaccharide portion of ganglioside GT1a. FIG. 1C: LOS of C. jejuniOH4382 has structural similarity to the oligosaccharide portion ofganglioside GD3.

FIGS. 2A–2B show the genetic organization of the cst-I locus from OH4384and comparison of the LOS biosynthesis loci from OH4384 and NCTC 11168.The distance between the scale marks is 1 kb. FIG. 2A shows a schematicrepresentation of the OH4384 cst-I locus, based on the nucleotidesequence which is available from GenBank (#AF130466). The partial prfBgene is somewhat similar to a peptide chain release factor (GenBank#AE000537) from Helicobacter pylori, while the cysD gene and the partialcysN gene are similar to E. coli genes encoding sulfateadenylyltransferase subunits (GenBank #AE000358). FIG. 2B shows aschematic representation of the OH4384 LOS biosynthesis locus, which isbased on the nucleotide sequence from GenBank (#AF130984). Thenucleotide sequence of the OH4382 LOS biosynthesis locus is identical tothat of OH4384 except for the cgtA gene, which is missing an “A” (seetext and GenBank #AF167345). The sequence of the NCTC 11168 LOSbiosynthesis locus is available from the Sanger Centre. Correspondinghomologous genes have the same number with a trailing “a” for the OH4384genes and a trailing “b” for the NCTC 11168 genes. A gene unique to theOH4384 strain is shown in black and genes unique to NCTC 11168 are shownin grey. The OH4384 ORF's #5a and #10a are found as an in-frame fusionORF (#5b/10b) in NCTC 11168 and are denoted with an asterisk (*).Proposed-functions for each ORF are found in Table 4.

FIG. 3 shows an alignment of the deduced amino acid sequences for thesialyltransferases. The OH4384 cst-I gene (SEQ ID NO:48, first 300residues), OH4384 cst-II gene (SEQ ID NO:3, identical to OH4382 cst-II),O:19 (serostrain) cst-II gene (SEQ ID NO:9, GenBank #AF167344), NCTC11168 cst-II gene (SEQ ID NO: 10) and an H. influenzae putative ORF (SEQID NO:49, GenBank #U32720) were aligned using the ClustalX alignmentprogram (Thompson et al. (1997) Nucleic Acids Res. 25, 4876–82). Theshading was produced by the program GeneDoc (Nicholas, K. B., andNicholas, H. B. (1997).

FIG. 4 shows a scheme for the enzymatic synthesis of ganglioside mimicsusing C. jejuni OH4384 glycosyltransferases. Starting from a syntheticacceptor molecule, a series of ganglioside mimics was synthesized withrecombinant α-2,3-sialyltransferase (Cst-I),β-1,4-N-acetylgalactosaminyltransferase (CgtA),β-1,3-galactosyltransferase (CgtB), and a bi-functionalα-2,3/α-2,8-sialyltransferase (Cst-II) using the sequences shown. Allthe products were analyzed by mass spectrometry and the observedmonoisotopic masses (shown in parentheses) were all within 0.02% of thetheoretical masses. The GM3, GD3, GM2 and GM1a mimics were also analyzedby NMR spectroscopy (see Table 4).

SUMMARY OF THE INVENTION

The present invention provides prokaryotic glycosyltransferase enzymesand nucleic acids that encode the enzymes. In one embodiment, theinvention provides isolated and/or recombinant nucleic acid moleculesthat include a polynucleotide sequence that encodes a polypeptideselected from the group consisting of:

-   -   a) a polypeptide having lipid A biosynthesis acyltransferase        activity, wherein the polypeptide comprises an amino acid        sequence that is at least about 70% identical to an amino acid        sequence encoded by nucleotides 350–1234 (ORF 2a) of the LOS        biosynthesis locus of C. jejuni strain OH4384 as shown in SEQ ID        NO:1;    -   b) a polypeptide having glycosyltransferase activity, wherein        the polypeptide comprises an amino acid sequence that is at        least about 70% identical to an amino acid sequence encoded by        nucleotides 1234–2487 (ORF 3a) of the LOS biosynthesis locus        of C. jejuni strain OH4384 as shown in SEQ ID NO:1;    -   c) a polypeptide having glycosyltransferase activity, wherein        the polypeptide comprises an amino acid sequence that is at        least about 50% identical to an amino acid sequence encoded by        nucleotides 2786–3952 (ORF 4a) of the LOS biosynthesis locus        of C. jejuni strain OH4384 as shown in SEQ ID NO:1 over a region        at least about 100 amino acids in length;    -   d) a polypeptide having β1,4-GalNAc transferase activity,        wherein the GalNAc transferase polypeptide has an amino acid        sequence that is at least about 77% identical to an amino acid        sequence as set forth in SEQ ID NO:17 over a region at least        about 50 amino acids in length;    -   e) a polypeptide having β1,3-galactosyltransferase activity,        wherein the galactosyltransferase polypeptide has an amino acid        sequence that is at least about 75% identical to an amino acid        sequence as set forth in SEQ ID NO:27 or SEQ ID NO:29 over a        region at least about 50 amino acids in length;    -   f) a polypeptide having either α2,3 sialyltransferase activity        or both α2,3- and α2,8 sialyltransferase activity, wherein the        polypeptide has an amino acid sequence that is at least about        66% identical over a region at least about 60 amino acids in        length to an amino acid sequence as set forth in one or more of        SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:10;    -   g) a polypeptide having sialic acid synthase activity, wherein        the polypeptide comprises an amino acid sequence that is at        least about 70% identical to an amino acid sequence encoded by        nucleotides 6924–7961 of the LOS biosynthesis locus of C. jejuni        strain OH4384 as shown in SEQ ID NO:1;    -   h) a polypeptide having sialic acid biosynthesis activity,        wherein the polypeptide comprises an amino acid sequence that is        at least about 70% identical to an amino acid sequence encoded        by nucleotides 8021–9076 of the LOS biosynthesis locus of C.        jejuni strain OH4384 as shown in SEQ ID NO:1;    -   i) a polypeptide having CNP-sialic acid synthetase activity,        wherein the polypeptide comprises an amino acid sequence that is        at least about 65% identical to an amino acid sequence encoded        by nucleotides 9076–9738 of the LOS biosynthesis locus of C.        jejuni strain OH4384 as shown in SEQ ID NO:1;    -   j) a polypeptide having acetyltransferase activity, wherein the        polypeptide comprises an amino acid sequence that is at least        about 65% identical to an amino acid sequence-encoded by        nucleotides 9729–10559 of the LOS biosynthesis locus of C.        jejuni strain OH4384 as shown in SEQ ID NO:1; and    -   k) a polypeptide having glycosyltransferase activity, wherein        the polypeptide comprises an amino acid sequence that is at        least about 65% identical to an amino acid sequence encoded by a        reverse complement of nucleotides 10557–11366 of the LOS        biosynthesis locus of C. jejuni strain OH4384 as shown in SEQ ID        NO:1.

In presently preferred embodiments, the invention provides an isolatednucleic acid molecule that includes a polynucleotide sequence thatencodes one or more polypeptides selected from the group consisting of:a) a sialyltransferase polypeptide that has both an α2,3sialyltransferase activity and an α2,8 sialyltransferase activity,wherein the sialyltransferase polypeptide has an amino acid sequencethat is at least about 76% identical to an amino acid sequence as setforth in SEQ ID NO:3 over a region at least about 60 amino acids inlength; b) a GalNAc transferase polypeptide that has a β1,4-GalNActransferase activity, wherein the GalNAc transferase polypeptide has anamino acid sequence that is at least about 75% identical to an aminoacid sequence as set forth in SEQ ID NO:17 over a region at least about50 amino acids in length; and c) a galactosyltransferase polypeptidethat has β1,3-galactosyltransferase activity, wherein thegalactosyltransferase polypeptide has an amino acid sequence that is atleast about 75% identical to an amino acid sequence as set forth in SEQID NO:27 over a region at least about 50 amino acids in length.

Also provided by the invention are expression cassettes and expressionvectors in which a glycosyltransferase nucleic acid of the invention isoperably linked to a promoter and other control sequences thatfacilitate expression of the glycosyltransferases in a desired hostcell. Recombinant host cells that express the glycosyltransferases ofthe invention are also provided.

The invention also provides isolated and/or recombinantly producedpolypeptides selected from the group consisting of:

-   -   a) a polypeptide having lipid A biosynthesis acyltransferase        activity, wherein the polypeptide comprises an amino acid        sequence that is at least about 70% identical to an amino acid        sequence encoded by nucleotides 350–1234 (ORF 2a) of the LOS        biosynthesis locus of C. jejuni strain OH4384 as shown in SEQ ID        NO:1;    -   b) a polypeptide having glycosyltransferase activity, wherein        the polypeptide comprises an amino acid sequence that is at        least about 70% identical to an amino acid sequence encoded by        nucleotides 1234–2487 (ORF 3a) of the LOS biosynthesis locus        of C. jejuni strain OH4384 as shown in SEQ ID NO:1;    -   c) a polypeptide having glycosyltransferase activity, wherein        the polypeptide comprises an amino acid sequence that is at        least about 50% identical to an amino acid sequence encoded by        nucleotides 2786–3952 (ORF 4a) of the LOS biosynthesis locus        of C. jejuni strain OH4384 as shown in SEQ ID NO:1 over a region        at least about 100 amino acids in length;    -   d) a polypeptide having β1,4-GalNAc transferase activity,        wherein the GalNAc transferase polypeptide has an amino acid        sequence that is at least about 77% identical to an amino acid        sequence as set forth in SEQ ID NO:17 over a region at least        about 50 amino acids in length;    -   e) a polypeptide having β1,3-galactosyltransferase activity,        wherein the galactosyltransferase polypeptide has an amino acid        sequence that is at least about 75% identical to an amino acid        sequence as set forth in SEQ ID NO:27 or SEQ ID NO:29 over a        region at least about 50 amino acids in length;    -   f) a polypeptide having either α2,3 sialyltransferase activity        or both α2,3 and α2,8 sialyltransferase activity, wherein the        polypeptide has an amino acid sequence that is at least about        66% identical to an amino acid sequence as set forth in SEQ ID        NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:10 over a region at        least about 60 amino acids in length;    -   g) a polypeptide having sialic acid synthase activity, wherein        the polypeptide comprises an amino acid sequence that is at        least about 70% identical to an amino acid sequence encoded by        nucleotides 6924–7961 of the LOS biosynthesis locus of C. jejuni        strain OH4384 as shown in SEQ ID NO:1;    -   h) a polypeptide having sialic acid biosynthesis activity,        wherein the polypeptide comprises an amino acid sequence that is        at least about 70% identical to an amino acid sequence encoded        by nucleotides 8021–9076 of the LOS biosynthesis locus of C.        jejuni strain OH4384 as shown in SEQ ID NO:1;    -   i) a polypeptide having CMP-sialic acid synthetase activity        wherein the polypeptide comprises an amino acid sequence that is        at least about 65% identical to an amino acid sequence encoded        by nucleotides 9076–9738 of the LOS biosynthesis locus of C.        jejuni strain OH4384 as shown in SEQ ID NO:1;    -   j) a polypeptide having acetyltransferase activity, wherein the        polypeptide comprises an amino acid sequence that is at least        about 65% identical to an amino acid sequence encoded by        nucleotides 9729–10559 of the LOS biosynthesis locus of C.        jejuni strain OH4384 as shown in SEQ ID NO:1; and    -   k) a polypeptide having glycosyltransferase activity, wherein        the polypeptide comprises an amino acid sequence that is at        least about 65% identical to an amino acid sequence encoded by a        reverse complement of nucleotides 10557–11366 of the LOS        biosynthesis locus of C. jejuni strain OH4384 as shown in SEQ ID        NO:1.

In presently preferred embodiments, the invention providesglycosyltransferase polypeptides including: a) a sialyltransferasepolypeptide that has both an α2,3 sialyltransferase activity and an α2.Ssialyltransferase activity, wherein the sialyltransferase polypeptidehas an amino acid sequence that is at least about 76% identical to anamino acid sequence as set forth in SEQ ID NO:3 over a region at leastabout 60 amino acids in length; b) a GalNAc transferase polypeptide thathas a β1,4-GalNAc transferase activity, wherein the GalNAc transferasepolypeptide has an amino acid sequence that is at least about 75%identical to an amino acid sequence as set forth in SEQ ID NO:17, over aregion at least about 50 amino acids in length; and c) agalactosyltransferase polypeptide that has β1,3-galactosyltransferaseactivity, wherein the galactosyltransferase polypeptide has an aminoacid sequence that is at least about 75% identical to an amino acidsequence as set forth in SEQ ID NO:27 or SEQ ID NO:29 over a region atleast about 50 amino acids in length.

The invention also provides reaction mixtures for the synthesis of asialylated oligosaccharide. The reaction mixtures include asialyltransferase polypeptide which has both an α2,3 sialyltransferaseactivity and an α2,8 sialyltransferase activity. Also present in thereaction mixtures are a galactosylated acceptor moiety and asialyl-nucleotide sugar. The sialyltransferase transfers a first sialicacid residue from the sialyl-nucleotide sugar (e.g., CMP-sialic acid) tothe galactosylated acceptor moiety in an α2,3 linkage, and further addsa second sialic acid residue to the first sialic acid residue in an α2,8linkage.

In another embodiment, the invention provides methods for synthesizing asialylated oligosaccharide. These methods involve incubating a reactionmixture that includes a sialyltransferase polypeptide which has both anα2,3 sialyltransferase activity and an α2,8 sialyltransferase activity,a galactosylated acceptor moiety, and a sialyl-nucleotide sugar, undersuitable conditions wherein the sialyltransferase polypeptide transfersa first sialic acid residue from the sialyl-nucleotide sugar to thegalactosylated acceptor moiety in an α2,3 linkage, and further transfersa second sialic acid residue to the first sialic acid residue in an α2,8linkage.

DETAILED DESCRIPTION

Definitions

The glycosyltransferases, reaction mixtures, and methods of theinvention are useful for transferring a monosaccharide from a donorsubstrate to an acceptor molecule. The addition generally takes place atthe non-reducing end of an oligosaccharide or carbohydrate moiety on abiomolecule. Biomolecules as defined here include, but are not limitedto, biologically significant molecules such as carbohydrates, proteins(e.g., glycoproteins), and lipids (e.g., glycolipids, phospholipids,sphingolipids and gangliosides).

The following abbreviations are used herein:

Ara=arabinosyl;

Fru=fructosyl;

Fuc=fucosyl;

Gal=galactosyl;

GalNAc=N-acetylgalactosaminyl;

Glc=glucosyl;

GlcNAc=N-acetylglucosaminyl;

Man=mannosyl; and

NeuAc=sialyl(N-acetylneuraminyl).

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid(2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550–11557; Kanarnon et al. (1990) J. Biol. Chem. 265:21811–21819. Also included are 9-substituted sialic acids such as a9-O-C₁–C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki (1992) Glycobiology 2: 25–40;Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992); Schauer, Methods in Enzymology, 50:64–89 (1987), and Schaur, Advances in Carbohydrate Chemistry andBiochemistry, 40: 131–234.The synthesis and use of sialic acid compoundsin a sialylation procedure is disclosed in international application WO92/16640, published Oct. 1, 1992.

Donor substrates for glycosyltransferases are activated nucleotidesugars. Such activated sugars generally consist of uridine and guanosinediphosphates, and cytidine monophosphate derivatives of the sugars inwhich the nucleoside diphosphate or monophosphate serves as a leavinggroup. Bacterial, plant, and fungal systems can sometimes use otheractivated nucleotide sugars.

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right.

All oligosaccharides described herein are described with the name orabbreviation for the non-reducing saccharide (e.g., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond, thering position of the reducing saccharide involved in the bond, and thenthe name or abbreviation of the reducing saccharide (e.g., GlcNAc). Thelinkage between two sugars may be expressed, for example, as 2,3, 2→3,or (2,3). Each saccharide is a pyranose or furanose.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues of naturalnucleotides that hybridize to nucleic acids in manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence includes the complementary sequencethereof.

The term “operably linked” refers to functional linkage between anucleic acid expression control sequence (such as a promoter, signalsequence, or array of transcription factor binding sites) and a secondnucleic acid sequence, wherein the expression control sequence affectstranscription and/or translation of the nucleic acid corresponding tothe second sequence.

A “heterologous polynucleotide” or a “heterologous nucleic acid”, asused herein, is one that originates from a source foreign to theparticular host cell, or, if from the same source, is modified from itsoriginal form. Thus, a heterologous glycosyltransferase gene in a hostcell includes a glycosyltransferase gene that is endogenous to theparticular host cell but has been modified. Modification of theheterologous sequence may occur, e.g., by treating the DNA with arestriction enzyme to generate a DNA fragment that is capable of beingoperably linked to a promoter. Techniques such as site-directedmutagenesis are also useful for modifying a heterologous sequence.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, or expresses a peptideor protein encoded by a heterologous nucleic acid. Recombinant cells cancontain genes that are not found within the native (non-recombinant)form of the cell. Recombinant cells also include those that containgenes that are found in the native form of the cell, but are modifiedand re-introduced into the cell by artificial means. The term alsoencompasses cells that contain a nucleic acid endogenous to the cellthat has been modified without removing the nucleic acid from the cell;such modifications include those obtained by gene replacement,site-specific mutation, and related techniques known to those of skillin the art.

A “recombinant nucleic acid” is a nucleic acid that is in a form that isaltered from its natural state. For example, the term “recombinantnucleic acid” includes a coding region that is operably linked to apromoter and/or other expression control region, processing signal,another coding region, and the like, to which the nucleic acid is notlinked in its naturally occurring form. A “recombinant nucleic acid”also includes, for example, a coding region or other nucleic acid inwhich one or more nucleotides have been substituted, deleted, inserted,compared to the corresponding naturally occurring nucleic acid. Themodifications include those introduced by in vitro manipulation, in vivomodification, synthesis methods, and the like.

A “recombinantly produced polypeptide” is a polypeptide that is encodedby a recombinant and/or heterologous nucleic acid. For example, apolypeptide that is expressed from a C. jejuniglycosyltransferase-encoding nucleic acid which is introduced into E.coli is a “recombinantly produced polypeptide.” A protein expressed froma nucleic acid that is operably linked to a non-native promoter is oneexample of a “recombinantly produced polypeptide. Recombinantly producedpolypeptides of the invention can be used to synthesize gangliosides andother oligosaccharides in their unpurified form (e.g., as a cell lysateor an intact cell), or after being completely or partially purified.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of affecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes include at least promoters and optionally, transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used as described herein.For example, an expression cassette can also include nucleotidesequences that encode a signal sequence that directs secretion of anexpressed protein from the host cell. Transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

A “subsequence” refers to a sequence of nucleic acids or amino acidsthat comprise a part of a longer sequence of nucleic acids or aminoacids (e.g., polypeptide) respectively.

The term “isolated” is meant to refer to material that is substantiallyor essentially free from components which normally accompany thematerial as found in its native state. Typically, isolated proteins ornucleic acids of the invention are at least about 80% pure, usually atleast about 90%, and preferably at least about 95% pure. Purity orhomogeneity can be indicated by a number of means well known in the art,such as agarose or polyacrylarnide gel electrophoresis of a protein ornucleic acid sample, followed by visualization upon staining. Forcertain purposes high resolution will be needed and HPLC or a similarmeans for purification utilized. An “isolated” enzyme, for example, isone which is substantially or essentially free from components whichinterfere with the activity of the enzyme. An “isolated nucleic acid”includes, for example, one that is not present in the chromosome of thecell in which the nucleic acid naturally occurs.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80%, most preferably 90–95%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In a mostpreferred embodiment, the sequences are substantially identical over theentire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403–410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389–3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. For example, the comparisons can be performed using aBLASTN Version 2.0 algorithm with a wordlength (W) of 11, G=5, E=2,q=−2, and r=1, and a comparison of both strands. For amino acidsequences, the BLASTP Version 2.0 algorithm can be used, with thedefault values of wordlength (W) of 3, G=11, E=1, and a BLOSUM62substitution matrix. (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873–5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

The phrase “hybridizing specifically to”, refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. The term “stringentconditions” refers to conditions under which a probe will hybridize toits target subsequence, but to no other sequences. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium. (As the target sequences are generally presentin excess, at Tm, 50% of the probes are occupied at equilibrium).Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

The phrases “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind preferentially to a particular protein and do not bind in asignificant amount to other proteins present in the sample. Specificbinding to a protein under such conditions requires an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

“Conservatively modified variations” of a particular polynucleotidesequence refers to those polynucleotides that encode identical oressentially identical amino acid sequences, or where the polynucleotidedoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Every polynucleotidesequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

Furthermore, one of skill will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 5%, moretypically less than 1%) in an encoded sequence are “conservativelymodified variations” where the alterations result in the substitution ofan amino acid with a chemically similar amino acid. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. One of skill will appreciate that many conservativevariations of the fusion proteins and nucleic acid which encode thefusion proteins yield essentially identical products. For example, dueto the degeneracy of the genetic code, “silent substitutions” (i.e.,substitutions of a nucleic acid sequence which do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. As described herein,sequences are preferably optimized for expression in a particular hostcell used to produce the enzymes (e.g., yeast, human, and the like).Similarly, “conservative amino acid substitutions,” in one or a fewamino acids in an amino acid sequence are substituted with differentamino acids with highly similar properties (see, the definitionssection, supra), are also readily identified as being highly similar toa particular amino acid sequence, or to a particular nucleic acidsequence which encodes an amino acid. Such conservatively substitutedvariations of any particular sequence are a feature of the presentinvention. See also, Creighton (1984) Proteins, W.H. Freeman andCompany. In addition, individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids in an encoded sequence are also “conservatively modifiedvariations”.

Description of the Preferred Embodiments

The present invention provides novel glycosyltransferase enzymes, aswell as other enzymes that are involved in enzyme-catalyzedoligosaccharide synthesis. The glycosyltransferases of the inventioninclude sialyltransferases, including a bifunctional sialyltransferasethat has both an α2,3 and an α2,8 sialyltransferase activity. Alsoprovided are β1,3-galactosyltransferases, β1,4-GalNAc transferases,sialic acid synthases, CMP-sialic acid synthetases, acetyltransferases,and other glycosyltransferases. The enzymes of the invention areprokaryotic enzymes, include those involved in the biosynthesis oflipooligosaccharides (LOS) in various strains of Campylobacter jejuni.The invention also provides nucleic acids that encode these enzymes, aswell as expression cassettes and expression vectors for use inexpressing the glycosyltransferases. In additional embodiments, theinvention provides reaction mixtures and methods in which one or more ofthe enzymes is used to synthesize an oligosaccharide.

The glycosyltransferases of the invention are useful for severalpurposes. For example, the glycosyltransferases are useful as tools forthe chemo-enzymatic syntheses of oligosaccharides, includinggangliosides and other oligosaccharides that have biological activity.The glycosyltransferases of the invention, and nucleic acids that encodethe glycosyltransferases, are also useful for studies of thepathogenesis mechanisms of organisms that synthesize ganglioside mimics,such as C. jejuni. The nucleic acids can be used as probes, for example,to study expression of the genes involved in ganglioside mimeticsynthesis. Antibodies raised against the glycosyltransferases are alsouseful for analyzing the expression patterns of these genes that areinvolved in pathogenesis. The nucleic acids are also useful fordesigning antisense oligonucleotides for inhibiting expression of theCampylobacter enzymes that are involved in the biosynthesis ofganglioside mimics that can mask the pathogens from the host's immunesystem.

The glycosyltransferases of the invention provide several advantagesover previously available glycosyltransferases. Bacterialglycosyltransferases such as those of the invention can catalyze theformation of oligosaccharides that are identical to the correspondingmammalian structures. Moreover, bacterial enzymes are easier and lessexpensive to produce in quantity, compared to mammalianglycosyltransferases. Therefore, bacterial glycosyltransferases such asthose of the present invention are attractive replacements for mammalianglycosyltransferases, which can be difficult to obtain in large amounts.That the glycosyltransferases of the invention are of bacterial originfacilitates expression of large quantities of the enzymes usingrelatively inexpensive prokaryotic expression systems. Typically,prokaryotic systems for expression of polypeptide products involves amuch lower cost than expression of the polypeptides in mammalian cellculture systems.

Moreover, the novel bifunctional sialyltransferases of the inventionsimplify the enzymatic synthesis of biologically important molecules,such as gangliosides, that have a sialic acid attached by an α2,8linkage to a second sialic acid, which in turn is α2,3-linked to agalactosylated acceptor. While previous methods for synthesizing thesestructures required two separate sialyltransferases, only onesialyltransferase is required when the bifunctional sialyltransferase ofthe present invention is used. This avoids the costs associated withobtaining a second enzyme, and can also reduce the number of stepsinvolved in synthesizing these compounds.

A. Glycosyltransferases and Associated Enzymes

The present invention provides prokaryotic glycosyltransferasepolypeptides, as well as other enzymes that are involved in theglycosyltransferase-catalyzed synthesis of oligosaccharides, includinggangliosides and ganglioside mimics. In presently preferred embodiments,the polypeptides include those that are encoded by open reading frameswithin the lipooligosaccharide (LOS) locus of Campylobacter species(FIG. 1). Included among the enzymes of the invention areglycosyltransferases, such as sialyltransferases (including abifunctional sialyltransferase), β1,4-GalNAc transferases, andβ1,3-galactosyltransferases, among other enzymes as described herein.Also provided are accessory enzymes such as, for example, CMP-sialicacid synthetase, sialic acid synthase, acetyltransferase, anacyltransferase that is involved in lipid A biosynthesis, and an enzymeinvolved in sialic acid biosynthesis.

The glycosyltransferases and accessory polypeptides of the invention canbe purified from natural sources, e.g., prokaryotes such asCampylobacter species. In presently preferred embodiments, theglycosyltransferases are obtained from C. jejuni, in particular from C.jejuni serotype O:19, including the strains OH4384 and OH4382. Alsoprovided are glycosyltransferases and accessory enzymes obtained from C.jejuni serotypes O:10, O:41, and O:2. Methods by which theglycosyltransferase polypeptides can be purified include standardprotein purification methods including, for example, ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982) Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)).

In presently preferred embodiments, the glycosyltransferase andaccessory enzyme polypeptides of the invention are obtained byrecombinant expression using the glycosyltransferase- and accessoryenzyme-encoding nucleic acids described herein. Expression vectors andmethods for producing the glycosyltransferases are described in detailbelow.

In some embodiments, the glycosyltransferase polypeptides are isolatedfrom their natural milieu, whether recombinantly produced or purifiedfrom their natural cells. Substantially pure compositions of at leastabout 90 to 95% homogeneity are preferred for some applications, and 98to 99% or more homogeneity are most preferred. Once purified, partiallyor to homogeneity as desired, the polypeptides may then be used (e.g.,as immunogens for antibody production or for synthesis ofoligosaccharides, or other uses as described herein or apparent to thoseof skill in the art). The glycosyltransferases need not, however, beeven partially purified for use to synthesize a desired saccharidestructure. For example, the invention provides recombinantly producedenzymes that are expressed in a heterologous host cell and/or from arecombinant nucleic acid. Such enzymes of the invention can be used whenpresent in a cell lysate or an intact cell, as well as in purified form.

1. Sialyltransferases

In some embodiments, the invention provides sialyltransferasepolypeptides. The sialyltransferases have an α2,3-sialyltransferaseactivity, and in some cases also have an α2,8 sialyltransferaseactivity. These bifunctional sialyltransferases, when placed in areaction mixture with a suitable saccharide acceptor (e.g., a saccharidehaving a terminal galactose) and a sialic acid donor (e.g., CMP-sialicacid) can catalyze the transfer of a first sialic acid from the donor tothe acceptor in an α2,3 linkage. The sialyltransferase then catalyzesthe transfer of a second sialic acid from a sialic acid donor to thefirst sialic acid residue in an α2,8 linkage. This type ofSiaα2,8-Siaα2,3-Gal structure is often found in gangliosides, includingGD3 and GT1a as shown in FIG. 4.

Examples of bifunctional sialyltransferases of the invention are thosethat are found in Campylobacter species, such as C jejuni. A presentlypreferred bifunctional sialyltransferase of the invention is that of theC. jejuni serotype O:19. One example of a bifunctional sialyltransferaseis that of C. jejuni strain OH 4384; this sialyltransferase has an aminoacid sequence as shown in SEQ ID NO:3. Other bifunctionalsialyltransferases of the invention generally have an amino acidsequence that is at least about 76% identical to the amino acid sequenceof the C. jejuni OH4384 bifunctional sialyltransferase over a region atleast about 60 amino acids in length. More preferably, thesialyltransferases of the invention are at least about 85% identical tothe OH 4384 sialyltransferase amino acid sequence, and still morepreferably at least about 95% identical to the amino acid sequence ofSEQ ID NO:3, over a region of at least 60 amino acids in length. Inpresently preferred embodiments, the region of percent identity extendsover a region longer than 60 amino acids. For example, in more preferredembodiments, the region of similarity extends over a region of at leastabout 100 amino acids in length, more preferably a region of at leastabout 150 amino acids in length, and most preferably over the fulllength of the sialyltransferase. Accordingly, the bifunctionalsialyltransferases of the invention include polypeptides that haveeither or both the α2,3- and α2,8-sialyltransferase activity and are atleast about 65% identical, more preferably at least about 70% identical,more preferably at least about 80% identical, and most preferably atleast about 90% identical to the amino acid sequence of the C. jejuni OH4384 CstII sialyltransferase (SEQ ID NO:3) over a region of thepolypeptide that is required to retain the respective sialyltransferaseactivities. In some embodiments, the bifunctional sialyltransferases ofthe invention are identical to C. jejuni OH 4384 CstII sialyltransferaseover the entire length of the sialyltransferase.

The invention also provides sialyltransferases that have α2,3sialyltransferase activity, but little or no α2,8 sialyltransferaseactivity. For example, CstII sialyltransferase of the C. jejuni O:19serostrain (SEQ ID NO:9) differs from that of strain OH 4384 by eightamino acids, but nevertheless substantially lacks α2,8 sialyltransferaseactivity (FIG. 3). The corresponding sialyltransferase from the O:2serotype strain NCTC 11168 (SEQ ID NO:10) is 52% identical to that ofOH4384, and also has little or no α2,8-sialyltranfserase activity.Sialyltransferases that are substantially identical to the CstIIsialyltransferase of C. jejuni strain O:10 (SEQ ID NO:5) and O:41 (SEQID NO:7) are also provided. The sialyltransferases of the inventioninclude those that are at least about 65% identical, more preferably atleast about 70% identical, more preferably at least about 80% identical,and most preferably at least about 90% identical to the amino acidsequences of the C. jejuni O:10 (SEQ ID NO:5), O:41 (SEQ ID NO:7), O:19serostrain (SEQ ID NO:9), or O:2 serotype strain NCTC 11168 (SEQ IDNO:10). The sialyltransferases of the invention, in some embodiments,have an amino acid sequence that is identical to that of the O:10, O:41,O:19 serostrain or NCTC 11168 C. jejuni strains.

The percent identities can be determined by inspection, for example, orcan be determined using an alignment algorithm such as the BLASTPVersion 2.0 algorithm using the default parameters, such as a wordlength(W) of 3, G=11, E=1, and a BLOSUM62 substitution matrix.

Sialyltransferases of the invention can be identified, not only bysequence comparison, but also by preparing antibodies against the C.jejuni OH4384 bifunctional sialyltransferase, or othersialyltransferases provided herein, and determining whether theantibodies are specifically immunoreactive with a sialyltransferase ofinterest. To obtain a bifunctional sialyltransferase in particular, onecan identify an organism that is likely to produce a bifunctionalsialyltransferase by determining whether the organism displays both α2,3and α2,8-sialic acid linkages on its cell surfaces. Alternatively, or inaddition, one can simply do enzyme assays of an isolatedsialyltransferase to determine whether both sialyltransferase activitiesare present.

2. β1,4-GalNAc Transferase

The invention also provides β1,4-GalNAc transferase polypeptides (e.g.,CgtA). The β1,4-GalNAc transferases of the invention, when placed in areaction mixture, catalyze the transfer of a GalNAc residue from a donor(e.g., UDP-GalNAc) to a suitable acceptor saccharide (typically asaccharide that has a terminal galactose residue). The resultingstructure, GalNAcβ1,4-Gal-, is often found in gangliosides and othersphingoids, among many other saccharide compounds. For example, the CgtAtransferase can catalyze the conversion of the ganglioside GM3 to GM2(FIG. 4).

Examples of the β1,4-GalNAc transferases of the invention are those thatare produced by Campylobacter species, such as C. jejuni. One example ofa β1,4-GalNAc transferase polypeptide is that of C. jejuni strainOH4384, which has an amino acid sequence as shown in SEQ ID NO:17. Theβ1,4-GalNAc transferases of the invention generally include an aminoacid sequence that is at least about 75% identical to an amino acidsequence as set forth in SEQ ID NO:17 over a region at least about 50amino acids in length. More preferably, the β1,4-GalNAc transferases ofthe invention are at least about 85% identical to this amino acidsequence, and still more preferably are at least about 95% identical tothe amino acid sequence of SEQ ID NO:17, over a region of at least 50amino acids in length. In presently preferred embodiments, the region ofpercent identity extends over a longer region than 50 amino acids, morepreferably over a region of at least about 100 amino acids, and mostpreferably over the full length of the GalNAc transferase. Accordingly,the β1,4-GalNAc transferases of the invention include polypeptides thathave β1,4-GalNAc transferase activity and are at least about 65%identical, more preferably at least about 70% identical, more preferablyat least about 80% identical, and most preferably at least about 90%identical to the amino acid sequence of the C. jejuni OH 4384β1,4-GalNAc transferases (SEQ ID NO:17) over a region of the polypeptidethat is required to retain the β1,4-GalNAc transferase activity. In someembodiments, the β1,4-GalNAc transferases of the invention are identicalto C. jejuni OH 4384 β1,4-GalNAc transferase over the entire length ofthe β1,4-GalNAc transferase.

Again, the percent identities can be determined by inspection, forexample, or can be determined using an alignment algorithm such as theBLASTP Version 2.0 algorithm with a wordlength (W) of 3, G=11, E=1, anda BLOSUM62 substitution matrix.

One can also identify β1,4-GalNAc transferases of the invention byimmunoreactivity. For example, one can prepare antibodies against the C.jejuni OH4384 β1,4-GalNAc transferase of SEQ ID NO:17 and determinewhether the antibodies are specifically immunoreactive with aβ1,4-GalNAc transferase of interest.

3. β1,3-Galactosyltransferases

Also provided by the invention are β1,3-galactosyltransferases (CgtB).When placed in a suitable reaction medium, theβ1,3-galactosyltransferases of the invention catalyze the transfer of agalactose residue from a donor (e.g., UDP-Gal) to a suitable saccharideacceptor (e.g., saccharides having a terminal GalNAc residue). Among thereactions catalyzed by the β1,3-galactosyltransferases is the transferof a galactose residue to the oligosaccharide moiety of GM2 to form theGM1a oligosaccharide moiety.

Examples of the β1,3-galactosyltransferases of the invention are thoseproduced by Campylobacter species, such as C. jejuni. For example, oneβ1,3-galactosyl-transferase of the invention is that of C. jejuni strainOH4384, which has the amino acid sequence shown in SEQ ID NO:27.

Another example of a β1,3-galactosyltransferase of the invention is thatof the C. jejuni O:2 serotype strain NCTC 11168. The amino acid sequenceof this galactosyltransferase is set forth in SEQ ID NO:29. Thisgalactosyltransferase expresses well in E. coli, for example, andexhibits a high amount of soluble activity. Moreover, unlike the OH4384CgtB, which can add more than one galactose if a reaction mixturecontains an excess of donor and is incubated for a sufficiently longperiod of time, the NCTC 11168 β1,3-galactose does not have asignificant amount of polygalactosyltransferase activity. For someapplications, the polygalactosyltransferase activity of the OH4384enzyme is desirable, but in other applications such as synthesis of GM1mimics, addition of only one terminal galactose is desirable.

The β1,3-galactosyltransferases of the invention generally have an aminoacid sequence that is at least about 75% identical to an amino acidsequence of the OH 4384 or NCTC 11168.CgtB as set forth in SEQ ID NO:27and SEQ ID NO:29, respectively, over a region at least about 50 aminoacids in length. More preferably, the β1,3-galactosyltransferases of theinvention are at least about 85% identical to either of these amino acidsequences, and still more preferably are at least about 95% identical tothe amino acid sequences of SEQ ID NO:27 or SEQ ID NO:29, over a regionof at least 50 amino acids in length. In presently preferredembodiments, the region of percent identity extends over a longer regionthan 50 amino acids, more preferably over a region of at least about 100amino acids, and most preferably over the full length of thegalactosyltransferase. Accordingly, the β1,3-galactosyltransferases ofthe invention include polypeptides that have β1,3-galactosyltransferaseactivity and are at least about 65% identical, more preferably at leastabout 70% identical, more preferably at least about 80% identical, andmost preferably at least about 90% identical to the amino acid sequenceof the C. jejuni OH4384 β1,3-galactosyltransferase (SEQ ID NO:27) or theNCTC 11168 galactosyltransferase (SEQ ID NO:29) over a region of thepolypeptide that is required to retain the β1,3-galactosyltransferaseactivity. In some embodiments, the β1,3-galactosyltransferase of theinvention are identical to C. jejuni OH 4384 or NCTC 11168β1,3-galactosyltransferase over the entire length of theβ1,3-galactosyltransferase.

The percent identities can be determined by inspection, for example, orcan be determined using an alignment algorithm such as the BLASTPVersion 2.0 algorithm with a wordlength (W) of 3, G=11, E=1, and aBLOSUM62 substitution matrix.

The β1,3-galactosyltransferases of the invention can be obtained fromthe respective Campylobacter species, or can be produced recombinantly.One can identify the glycosyltransferases by assays of enzymaticactivity, for example, or by detecting specific immunoreactivity withantibodies raised against the C. jejuni OH4384β1,3-galactosyltransferase having an amino acid sequence as set forth inSEQ ID NO:27 or the C. jejuni NCTC 11168 β1,3 galactosyltransferase asset forth in SEQ ID NO:29.

4. Additional Enzymes Involved in LOS Biosynthetic Pathway

The present invention also provides additional enzymes that are involvedin the biosynthesis of oligosaccharides such as those found on bacteriallipooligosaccharides. For example, enzymes involved in the synthesis ofCMP-sialic acid, the donor for sialyltransferases, are provided. Asialic acid synthase is encoded by open reading frame (ORF) 8a of C.jejuni strain OH 4384 (SEQ ID NO:35) and by open reading frame 8b ofstrain NCTC 11168 (see, Table 3). Another enzyme involved in sialic acidsynthesis is encoded by ORF 9a of OH 4384 (SEQ ID NO:36) and 9b of NCTC11168. A CMP-sialic acid synthetase is encoded by ORF 10a (SEQ ID NO:37)and 10b of OH 4384 and NCTC 11168, respectively.

The invention also provides an acyltransferase that is involved in lipidA biosynthesis. This enzyme is encoded by open reading frame 2a of C.jejuni strain OH4384 (SEQ ID NO:32) and by open reading frame 2B ofstrain NCTC 11168. An acetyltransferase is also provided; this enzyme isencoded by ORF 11a of strain OH 4384 (SEQ ID NO:38); no homolog is foundin the LOS biosynthesis locus of strain NCTC 11168.

Also provided are three additional glycosyltransferases. These enzymesare encoded by ORFs 3a (SEQ ID NO:33), 4a (SEQ ID NO:34), and 12a (SEQID NO:39) of strain OH 4384 and ORFs 3b, 4b, and 12b of strain NCTC11168.

The invention includes, for each of these enzymes, polypeptidesthat-include an an amino acid sequence that is at least about 75%identical to an amino acid sequence as set forth herein over a region atleast about 50 amino acids in length. More preferably, the enzymes ofthe invention are at least about 85% identical to the respective aminoacid sequence, and still more preferably are at least about 95%identical to the amino acid sequence, over a region of at least 50 aminoacids in length. In presently preferred embodiments, the region ofpercent identity extends over a longer region than 50 amino acids, morepreferably over a region of at least about 100 amino acids, and mostpreferably over the full length of the enzyme. Accordingly, the enzymesof the invention include polypeptides that have the respective activityand are at least about 65% identical, more preferably at least about 70%identical, more preferably at least about 80% identical, and mostpreferably at least about 90% identical to the amino acid sequence ofthe corresponding enzyme as set forth herein over a region of thepolypeptide that is required to retain the respective enzymaticactivity. In some embodiments, the enzymes of the invention areidentical to the corresponding C. jejuni OH 4384 enzymes over the entirelength of the enzyme.

B. Nucleic Acids that Encode Glycosyltransferases and Related Enzymes

The present invention also provides isolated and/or recombinant nucleicacids that encode the glycosyltransferases and other enzymes of theinvention. The glycosyltransferase-encoding nucleic acids of theinvention are useful for several purposes, including the recombinantexpression of the corresponding glycosyltransferase polypeptides, and asprobes to identify nucleic acids that encode other glycosyltransferasesand to study regulation and expression of the enzymes.

Nucleic acids of the invention include those that encode an entireglycosyltransferase enzyme such as those described above, as well asthose that encode a subsequence of a glycosyltransferase polypeptide.For example, the invention includes nucleic acids that encode apolypeptide which is not a full-length glycosyltransferase enzyme, butnonetheless has glycosyltransferase activity. The nucleotide sequencesof the LOS locus of C. jejuni strain OH4384 is provided herein as SEQ IDNO:1, and the respective reading frames are identified. Additionalnucleotide sequences are also provided, as discussed below. Theinvention includes not only nucleic acids that include the nucleotidesequences as set forth herein, but also nucleic acids that aresubstantially identical to, or substantially complementary to, theexemplified embodiments. For example, the invention includes nucleicacids that include a nucleotide sequence that is at least about 70%identical to one that is set forth herein, more preferably at least 75%,still more preferably at least 80%, more preferably at least 85%, stillmore preferably at least 90%, and even more preferably at least about95% identical to an exemplified nucleotide sequence. The region ofidentity extends over at least about 50 nucleotides, more preferablyover at least about 100 nucleotides, still more preferably over at leastabout 500 nucleotides. The region of a specified percent identity, insome embodiments, encompasses the coding region of a sufficient portionof the encoded enzyme to retain the respective enzyme activity. Thespecified percent identity, in preferred embodiments, extends over thefull length of the coding region of the enzyme.

The nucleic acids that encode the glycosyltransferases of the inventioncan be obtained using methods that are known to those of skill in theart. Suitable nucleic acids (e.g., cDNA, genomic, or subsequences(probes)) can be cloned, or amplified by in vitro methods such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (SSR). A wide variety of cloning and invitro amplification methodologies are well-known to persons of skill.Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152Academic Press, Inc., San Diego, Calif. (Berger); Sanbrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1–3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.);Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashionet al., U.S. Pat. No. 5,017,478; and Carr, European Patent No.0,246,864. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al., eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C & EN 36–47; The Journal Of NIH Research (1991) 3:81–94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelliet al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989)J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science 241:1077–1080; Van Brunt (1990) Biotechnology 8: 291–294; Wu and Wallace(1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117. Improvedmethods of cloning in vitro amplified nucleic acids are described inWallace et al., U.S. Pat. No. 5,426,039.

Nucleic acids that encode the glycosyltransferase polypeptides of theinvention, or subsequences of these nucleic acids, can be prepared byany suitable method as described above, including, for example, cloningand restriction of appropriate sequences. As an example, one can obtaina nucleic-acid that encodes a glycosyltransferase of the invention byroutine cloning methods. A known nucleotide sequence of a gene thatencodes the glycosyltransferase of interest, such as are describedherein, can be used to provide probes that specifically hybridize to agene that encodes a suitable enzyme in a genomic DNA sample, or to amRNA in a total RNA sample (e.g., in a Southern or Northern blot).Preferably, the samples are obtained from prokaryotic organisms, such asCampylobacter species. Examples of Campylobacter species of particularinterest include C. jejuni. Many C. jejuni O:19 strains synthesizeganglioside mimics and are useful as a source of theglycosyltransferases of the invention.

Once the target glycosyltransferase nucleic acid is identified, it canbe isolated according to standard methods known to those of skill in theart (see, e.g., Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, 2nd Ed., Vols. 1–3, Cold Spring Harbor Laboratory; Berger andKimmel (1987) Methods in Enzymology, Vol. 152: Guide to MolecularCloning Techniques, San Diego: Academic Press, Inc.; or Ausubel et al.(1987) Current Protocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York).

A nucleic acid that encodes a glycosyltransferase of the invention canalso be cloned by detecting its expressed product by means of assaysbased on the physical, chemical, or immunological properties. Forexample, one can identify a cloned bifunctionalsialyltransferase-encoding nucleic acid by the ability of a polypeptideencoded by the nucleic acid to catalyze the coupling of a sialic acid inan α2,3-linkage to a galactosylated acceptor, followed by the couplingof a second sialic acid residue to the first sialic acid in an α2,8linkage. Similarly, one can identify a cloned nucleic acid that encodesa β1,4-GalNAc transferase or a β1,3-galactosyltransferase by the abilityof the encoded polypeptide to catalyze the transfer of a GalNAc residuefrom UDP-GalNAc, or a galactose residue from UDP-Gal, respectively, to asuitable acceptor. Suitable assay conditions are known in the art, andinclude those that are described in the Examples. Other physicalproperties of a polypeptide expressed from a particular nucleic acid canbe compared to properties of known glycosyltransferase polypeptides ofthe invention, such as those described herein, to provide another methodof identifying nucleic acids that encode glycosyltransferases of theinvention. Alternatively, a putative glycosyltransferase gene can bemutated; and its role as a glycosyltransferase established by detectinga variation in the ability to produce the respective glycoconjugate.

In other embodiments, glycosyltransferase-encoding nucleic acids can becloned using DNA amplification methods such as polymerase chain reaction(PCR). Thus, for example, the nucleic acid sequence or subsequence isPCR amplified, preferably using a sense primer containing onerestriction site (e.g., XbaI) and an antisense primer containing anotherrestriction site (e.g., HindIII). This will produce a nucleic acidencoding the desired glycosyltransferase amino acid sequence orsubsequence and having terminal restriction sites. This nucleic acid canthen be easily ligated into a vector containing a nucleic acid encodingthe second molecule and having the appropriate corresponding restrictionsites. Suitable PCR primers can be determined by one of skill in the artusing the sequence information provided herein. Appropriate restrictionsites can also be added to the nucleic acid encoding theglycosyltransferase of the invention, or amino acid subsequence, bysite-directed mutagenesis. The plasmid containing theglycosyltransferase-encoding nucleotide sequence or subsequence iscleaved with the appropriate restriction endonuclease and then ligatedinto an appropriate vector for amplification and/or expression accordingto standard methods.

Examples of suitable primers suitable for amplification of theglycosyltransferase-encoding nucleic acids of the invention are shown inTable 2; some of the primer pairs are designed to provide a 5′ NdeIrestriction site and a 3′ SalI site on the amplified fragment. Theplasmid containing the enzyme-encoding sequence or subsequence iscleaved with the appropriate restriction endonuclease and then ligatedinto an appropriate vector for amplification and/or expression accordingto standard methods.

As an alternative to cloning a glycosyltransferase-encoding nucleicacid, a suitable nucleic acid can be chemically synthesized from a knownsequence that encodes a glycosyltransferase of the invention. Directchemical synthesis methods include, for example, the phosphotriestermethod of Narang et al. (1979) Meth. Enzymol. 68: 90–99; thephosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109–151;the diethylphosphoramidite method of Beaucage et al. (1981) Tetra.Lett., 22: 1859–1862; and the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is often limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences. Alternatively, subsequences may be clonedand the appropriate subsequences cleaved using appropriate restrictionenzymes. The fragments can then be ligated to produce the desired DNAsequence.

In some embodiments, it may be desirable to modify the enzyme-encodingnucleic acids. One of skill will recognize many ways of generatingalterations in a given nucleic acid construct. Such well-known methodsinclude site-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques. See,e.g., Giliman and Smith (1979) Gene 8:81–97, Roberts et al. (1987)Nature 328: 731–734.

In a presently preferred embodiment, the recombinant nucleic acidspresent in the cells of the invention are modified to provide preferredcodons which enhance translation of the nucleic acid in a selectedorganism (e.g., E. coli preferred codons are substituted into a codingnucleic acid for expression in E. coli).

The present invention includes nucleic acids that are isolated (i.e.,not in their native chromosomal location) and/or recombinant (i.e.,modified from their original form, present in a non-native organism,etc.).

1. Sialyltransferases

The invention provides nucleic acids that encode sialyltransferases suchas those described above. In some embodiments, the nucleic acids of theinvention encode bifunctional sialyltransferase polypeptides that haveboth an α2,3 sialyltransferase activity and an α2,8 sialyltransferaseactivity. These sialyltransferase nucleic acids encode asialyltransferase polypeptide that has an amino acid sequence that is atleast about 76% identical to an amino acid sequence as set forth in SEQID NO:3 over a region at least about 60 amino acids in length. Morepreferably the sialyltransferases encoded by the nucleic acids of theinvention are at least about 85% identical to the amino acid sequence ofSEQ ID NO:3, and still more preferably at least about 95% identical tothe amino acid sequence of SEQ ID NO:3, over a region of at least 60amino acids in length. In presently preferred embodiments, the region ofpercent identity extends over a longer region than 60 amino acids, morepreferably over a region of at least about 100 amino acids, and mostpreferably over the full length of the sialyltransferase. In a presentlypreferred embodiment, the sialyltransferase-encoding nucleic acids ofthe invention encode a polypeptide having the amino acid sequence asshown in SEQ ID NO:3.

An example of a nucleic acid of the invention is an isolated and/orrecombinant form of a bifunctional sialyltransferase-encoding nucleicacid of C. jejuni OH4384. The nucleotide sequence of this nucleic acidis shown in SEQ ID NO:2. The sialyltransferase-encoding polynucleotidesequences of the invention are typically at least about 75% identical tothe nucleic acid sequence of SEQ ID NO:2 over a region at least about 50nucleotides in length. More preferably, the sialyltransferase-encodingnucleic acids of the invention are at least about 85% identical to thisnucleotide sequence, and still more preferably are at least about 95%identical to the nucleotide sequence of SEQ ID NO:2, over a region of atleast 50 amino acids in length. In presently preferred embodiments, theregion of the specified percent identity threshold extends over a longerregion than 50 nueleotides, more preferably over a region of at leastabout 100 nucleotides, and most preferably over the full length of thesialyltransferase-encoding region. Accordingly, the invention providesbifunctional sialyltransferase-encoding nucleic acids that aresubstantially identical to that of the C. jejuni strain OH4384 cstII asset forth in SEQ ID NO:2 or strain O:10 (SEQ ID NO:4).

Other sialyltransferase-encoding nucleic acids of the invention encodesialyltransferases have α2,3 sialyltransferase activity but lacksubstantial α2,8 sialyltransferase activity. For example, nucleic acidsthat encode a CstII α2,3 sialyltransferase from C. jejuni serostrainO:19 (SEQ ID NO:8) and NCTC 11168 are provided by the invention; theseenzymes have little or no α2,8-sialyltransferase activity (Table 6).

To identify nucleic acids of the invention, one can use visualinspection, or can use a suitable alignment algorithm. An alternativemethod by which one can identify a bifunctionalsialyltransferase-encoding nucleic acid of the invention is byhybridizing, under stringent conditions, the nucleic acid of interest toa nucleic acid that includes a polynucleotide sequence of asialyltransferase as set forth herein.

2. β1,4-GalNAc transferases

Also provided by the invention are nucleic acids that includepolynucleotide sequences that encode a GalNAc transferase polypeptidethat has a β1,4-GalNAc transferase activity. The polynucleotidesequences encode a GalNAc transferase polypeptide that has an amino acidsequence that is at least about 70% identical to the C. jejuni OH4384β1,4-GalNAc transferase, which has an amino acid sequence as set forthin SEQ ID NO:17, over a region at least about 50 amino acids in length.More preferably the GalNAc transferase polypeptide encoded by thenucleic acids of the invention are at least about 80% identical to thisamino acid sequence, and still more preferably at least about 90%identical to the amino acid sequence of SEQ ID NO:17, over a region ofat least 50 amino acids in length. In presently preferred embodiments,the region of percent identity extends over a longer region than 50amino acids, more preferably over a region of at least about 100 aminoacids, and most preferably over the full length of the GalNActransferase polypeptide. In a presently preferred embodiment, the GalNActransferase polypeptide-encoding nucleic acids of the invention encode apolypeptide having the amino acid sequence as shown in SEQ ID NO:17. Toidentify nucleic acids of the invention, one can use visual inspection,or can use a suitable alignment algorithm.

One example of a GalNAc transferase-encoding nucleic acid of theinvention is an isolated and/or recombinant form of the GalNActransferase-encoding nucleic acid of C. jejuni OH4384. This nucleic acidhas a nucleotide sequence as shown in SEQ ID NO:16. The GalNActransferase-encoding polynucleotide sequences of the invention aretypically at least about 75% identical to the nucleic acid sequence ofSEQ ID NO:16 over a region at least about 50 nucleotides in length. Morepreferably, the GalNAc transferase-encoding nucleic acids of theinvention are at least about 85% identical to this nucleotide sequence,and still more preferably are at least about 95% identical to thenucleotide sequence of SEQ ID NO:16, over a region of at least 50 aminoacids in length. In presently preferred embodiments, the region ofpercent identity extends over a longer region than 50 nucleotides, morepreferably over a region of at least about 100 nucleotides, and mostpreferably over the full length of the GalNAc transferase-encodingregion.

To identify nucleic acids of the invention, one can use visualinspection, or can use a suitable alignment algorithm. An alternativemethod-by which one can identify a GalNAc transferase-encoding nucleicacid of the invention is by hybridizing, under stringent conditions, thenucleic acid of interest to a nucleic acid that includes apolynucleotide sequence of SEQ ID NO:16.

3. β1,3-Galactosyltransferases

The invention also provides nucleic acids that include polynucleotidesequences that encode a polypeptide that has β1,3-galactosyltransferaseactivity (CgtB). The β1,3-galactosyltransferase polypeptides encoded bythese nucleic acids of the invention preferably include an amino acidsequence that is at least about 75% identical to an amino acid sequenceof a C. jejuni strain OH4384 β1,3-galactosyltransferase as set forth inSEQ ID NO:27, or to that of a strain NCTC 11168β1,3-galactosyltransferase as set forth in SEQ ID NO:29, over a regionat least about 50 amino acids in length. More preferably, thegalactosyltransferase polypeptides encoded by these nucleic acids of theinvention are at least about 85% identical to this amino acid sequence,and still more preferably are at least about 95% identical to the aminoacid sequence of SEQ ID NO:27 or SEQ ID NO:29, over a region of at least50 amino acids in length. In presently preferred embodiments, the regionof percent identity extends over a longer region than 50 amino acids,more preferably over a region of at least about 100 amino acids, andmost preferably over the full length of the galactosyltransferasepolypeptide-encoding region.

One example of a β1,3-galactosyltransferase-encoding nucleic acid of theinvention is an isolated and/or recombinant form of theβ1,3-galactosyltransferase-encoding nucleic acid of C. jejuni OH4384.This nucleic acid includes a nucleotide sequence as shown in SEQ IDNO:26. Another suitable β1,3-galactosyltransferase-encoding nucleic acidincludes a nucleotide sequence of a C. jejuni NCTC 11168 strain, forwhich the nucleotide sequence is shown in SEQ ID NO:28. Theβ1,3-galactosyltransferase-encoding polynucleotide sequences of theinvention are typically at least about 75% identical to the nucleic acidsequence of SEQ ID NO:26 or that of SEQ ID NO:28 over a region at leastabout 50 nucleotides in length. More preferably, theβ1,3-galactosyltransferase-encoding nucleic acids of the invention areat least about 85% identical to at least one of these nucleotidesequences, and still more preferably are at least about 95% identical tothe nucleotide sequences of SEQ ID NO:26 and/or SEQ ID NO:28, over aregion of at least 50 amino acids in length. In presently preferredembodiments, the region of percent identity extends over a longer regionthan 50 nucleotides, more preferably over a region of at least about 100nucleotides, and most preferably over the full length of theβ1,3-galactosyltransferase-encoding region.

To identify nucleic acids of the invention, one can use visualinspection, or can use a suitable alignment algorithm. An alternativemethod by which one can identify a galactosyltransferasepolypeptide-encoding nucleic acid of the invention is by hybridizing,under stringent conditions, the nucleic acid of interest to a nucleicacid that includes a polynucleotide sequence of SEQ ID NO:26 or SEQ IDNO:28.

4. Additional Enzymes Involved in LOS Biosynthetic Pathway

Also provided are nucleic acids that encode other enzymes that areinvolved in the LOS biosynthetic pathway of prokaryotes such asCampylobacter. These nucleic acids encode enzymes such as, for example,sialic acid synthase, which is encoded by open reading frame (ORF) 8a ofC. jejuni strain OH 4384 and by open reading frame 8b of strain NCTC11168 (see, Table 3), another enzyme involved in sialic acid synthesis,which is encoded by ORF 9a of OH 4384 and 9b of NCTC 11168, and aCMP-sialic acid synthetase which is encoded by ORF 10a and 10b of OH4384 and NCTC 11168, respectively.

The invention also provides nucleic acids that encode an acyltransferasethat is involved in lipid A biosynthesis. This enzyme is encoded by openreading frame 2a of C. jejuni strain OH4384 and by open reading frame 2Bof strain NCTC 11168. Nucleic acids that encode an acetyltransferase arealso provided; this enzyme is encoded by ORF 11a of strain OH 4384; nohomolog is found in the LOS biosynthesis locus of strain NCTC 11168.

Also provided are nucleic acids that encode three additionalglycosyltransferases. These enzymes are encoded by ORFs 3a, 4a, and 12aof strain OH 4384 and ORFs 3b, 4b, and 12b of strain NH 11168 (FIG. 1).

C. Expression Cassettes and Expression of the Glycosyltransferases

The present invention also provides expression cassettes, expressionvectors, and recombinant host cells that can be used to produce theglycosyltransferases and other enzymes of the invention. A typicalexpression cassette contains a promoter operably linked to a nucleicacid that encodes the glycosyltransferase or other enzyme of interest.The expression cassettes are typically included on expression vectorsthat are introduced into suitable host cells, preferably prokaryotichost cells. More than one glycosyltransferase polypeptide can beexpressed in a single host cell by placing multiple transcriptionalcassettes in a single expression vector, by constructing a gene thatencodes a fusion protein consisting of more than oneglycosyltransferase, or by utilizing different expression vectors foreach glycosyltransferase.

In a preferred embodiment, the expression cassettes are useful forexpression of the glycosyltransferases in prokaryotic host cells.Commonly used prokaryotic control sequences, which are defined herein toinclude promoters for transcription initiation, optionally with anoperator, along with ribosome binding site sequences, include suchcommonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056),the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad.Sci. U.S.A. (1983) 80:21–25); and the lambda-derived P_(L) promoter andN-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128).The particular promoter system is not critical to the invention, anyavailable promoter that functions in prokaryotes can be used.

Either constitutive or regulated promoters can be used in the presentinvention. Regulated promoters can be advantageous because the hostcells can be grown to high densities before expression of theglycosyltransferase polypeptides is induced. High level expression ofheterologous proteins slows cell growth in some situations. Regulatedpromoters especially suitable for use in E. coli include thebacteriophage lambda P_(L) promoter, the hybrid trp-lac promoter (Amannet al., Gene (1983) 25: 167; de Boer et al., Proc. Natl. Acad. Sci. USA(1983) 80: 21, and the bacteriophage T7 promoter (Studier et al., J.Mol. Biol. (1986); Tabor et al., (1985). These promoters and their useare discussed in Sambrook et al., supra. A presently preferred regulablepromoter is the dual tac-gal promoter, which is described inPCT/US97/20528 (Int'l. Publ. No. WO 9820111).

For expression of glycosyltransferase polypeptides in prokaryotic cellsother than E. coli, a promoter that functions in the particularprokaryotic species is required. Such promoters can be obtained fromgenes that have been cloned from the species, or heterologous promoterscan be used. For example, a hybrid trp-lac promoter functions inBacillus in addition to E. coli. Promoters suitable for use ineukaryotic host cells are well known to those of skill in the art.

A ribosome binding site (RBS) is conveniently included in the expressioncassettes of the invention that are intended for use in prokaryotic hostcells. An RBS in E. coli, for example, consists of a nucleotide sequence3–9 nucleotides in length located 3–11 nucleotides upstream of theinitiation codon (Shine and Dalgarno, Nature (1975) 254: 34; Steitz, InBiological regulation and development: Gene expression (ed. R. F.Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, NY).

Translational coupling can be used to enhance expression. The strategyuses a short upstream open reading frame derived from a highly expressedgene native to the translational system, which is placed downstream ofthe promoter, and a ribosome binding site followed after a few aminoacid codons by a termination codon. Just prior to the termination codonis a second ribosome binding site, and following the termination codonis a start codon for the initiation of translation. The system dissolvessecondary structure in the RNA, allowing for the efficient initiation oftranslation. See Squires et. al. (1988) J. Biol. Chem. 263: 16297–16302.

The glycosyltransferase polypeptides of the invention can be expressedintracellularly, or can be secreted from the cell. Intracellularexpression often results in high yields. If necessary, the amount ofsoluble, active glycosyltransferase polypeptides can be increased byperforming refolding procedures (see, e.g., Sambrook et al., supra.;Marston et al., Bio/Technology (1984) 2: 800; Schoner et al.,Bio/Technology (1985) 3: 151). In embodiments in which theglycosyltransferase polypeptides are secreted from the cell, either intothe periplasm or into the extracellular medium, the polynucleotidesequence that encodes the glycosyltransferase is linked to apolynucleotide sequence that encodes a cleavable signal peptidesequence. The signal sequence directs translocation of theglycosyltransferase polypeptide through the cell membrane. An example ofa suitable vector for use in E. coli that contains a promoter-signalsequence unit is pTA1529, which has the E. coli phoA promoter and signalsequence (see, e.g., Sambrook et al., supra.; Oka et al., Proc. Natl.Acad. Sci. USA (1985) 82: 7212; Talmadge et al., Proc. Natl. Acad. Sci.USA (1980) 77: 3988; Takahara et al., J. Biol. Chem. (1985) 260: 2670).

The glycosyltransferase polypeptides of the invention can also beproduced as fusion proteins. This approach often results in high yields,because normal prokaryotic control sequences direct transcription andtranslation. In E. coli, lacZ fusions are often used to expressheterologous proteins. Suitable vectors are readily available, such asthe pUR, pEX, and pMR100 series (see, e.g., Sambrook et al., supra.).For certain applications, it may be desirable to cleave thenon-glycosyltransferase amino acids from the fusion protein afterpurification. This can be accomplished by any of several methods knownin the art, including cleavage by cyanogen bromide, a protease, or byFactor X_(a) (see, e.g., Sambrook et al., supra.; Itakura et al.,Science (1977) 198: 1056; Goeddel et al., Proc. Natl. Acad. Sci. USA(1979) 76: 106; Nagai et al., Nature (1984) 309: 810; Sung et al., Proc.Natl. Acad. Sci. USA (1986) 83: 561). Cleavage sites can be engineeredinto the gene for the fusion protein at the desired point of cleavage.

A suitable system for obtaining recombinant proteins from E. coli whichmaintains the integrity of their N-termini has been described by Milleret al. Biotechnology 7:698–704 (1989). In this system, the gene ofinterest is produced as a C-terminal fusion to the first 76 residues ofthe yeast ubiquitin gene containing a peptidase cleavage site. Cleavageat the junction of the two moieties results in production of a proteinhaving an intact authentic N-terminal residue.

Glycosyltransferases of the invention can be expressed in a variety ofhost cells, including E. coli, other bacterial hosts, yeast, and varioushigher eukaryotic cells such as the COS, CHO and HeLa cells lines andmyeloma-cell lines. Examples of useful bacteria include, but are notlimited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus,Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella,Rhizobia, Vitreoscilla, and Paracoccus. The recombinantglycosyltransferase-encoding nucleic acid is operably linked toappropriate expression control sequences for each host. For E. coli thisincludes a promoter such as the T7, trp, or lambda promoters, a ribosomebinding site and preferably a transcription termination signal. Foreukaryotic cells, the control sequences will include a promoter andpreferably an enhancer derived from immunoglobulin genes, SV40,cytomegalovirus, etc., and a polyadenylation sequence, and may includesplice donor and acceptor sequences.

The expression vectors of the invention can be transferred into thechosen host cell by well-known methods such as calcium chloridetransformation for E. coli and calcium phosphate treatment orelectroporation for mammalian cells. Cells transformed by the plasmidscan be selected by resistance to antibiotics conferred by genescontained on the plasmids, such as the amp, got, neo and hyg genes.

Once expressed, the recombinant glycosyltransferase polypeptides can bepurified according to standard procedures of the art, including ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred. Once purified, partially or to homogeneity as desired,the polypeptides may then be used (e.g., as immunogens for antibodyproduction). The glycosyltransferases can also be used in an unpurifiedor semi-purified state. For example, a host cell that expresses theglycosyltransferase can be used directly in a glycosyltransferasereaction, either with or without processing such as permeabilization orother cellular disruption.

One of skill would recognize that modifications can be made to theglycosyltransferase proteins without diminishing their biologicalactivity. Some modifications may be made to facilitate the cloning,expression, or incorporation of the targeting molecule into a fusionprotein. Such modifications are well known to those of skill in the artand include, for example, a methionine added at the amino terminus toprovide an initiation site, or additional amino acids (e.g., poly His)placed on either terminus to create conveniently located restrictionsites or termination codons or purification sequences.

D. Methods and Reaction Mixtures for Synthesis of Oligosaccharides

The invention provides reaction mixtures and methods in which theglycosyltransferases of the invention are used to prepare desiredoligosaccharides (which are composed of two or more saccharides). Theglycosyltransferase reactions of the invention take place in a reactionmedium comprising at least one glycosyltransferase, a donor substrate,an acceptor sugar and typically a soluble divalent metal cation. Themethods rely on the use of the glycosyltransferase to catalyze theaddition of a saccharide to a substrate (also referred to as an“acceptor”) saccharide. A number of methods of usingglycosyltransferases to synthesize desired oligosaccharide structuresare known. Exemplary methods are described, for instance, WO 96/32491,Ito et al. (1993) Pure Appl. Chem. 65:753, and U.S. Pat. Nos. 5,352,670,5,374,541, and 5,545,553.

For example, the invention provides methods for adding sialic acid in anα2,3 linkage to a galactose residue, by contacting a reaction mixturecomprising an activated sialic acid (e.g., CMP-NeuAc, CMP-NeuGc, and thelike) to an acceptor moiety that includes a terminal galactose residuein the presence of a bifunctional sialyltransferase of the invention. Inpresently preferred embodiments, the methods also result in the additionof a second sialic acid residue which is linked to the first sialic acidby an α2,8 linkage. The product of this method is Siaα2,8-Siaα2,3-Gal-.Examples of suitable acceptors include a terminal Gal that is linked toGlcNAc or Glc by a β1,4 linkage, and a terminal Gal that is β1,3-linkedto either GlcNAc or GalNAc. The terminal residue to which the sialicacid is attached can itself be attached to, for example, H, asaccharide, oligosaccharide, or an aglycone group having at least onecarbohydrate atom. In some embodiments, the acceptor residue is aportion of an oligosaccharide that is attached to a protein, lipid, orproteoglycan, for example.

In some embodiments, the invention provides reaction mixtures andmethods for synthesis of gangliosides, lysogangliosides, gangliosidemimics, lysoganglioside mimics, or the carbohydrate portions of thesemolecules. These methods and reaction mixtures-typically include as thegalactosylated acceptor moiety a compound having a formula selected fromthe group consisting of Gal4Glc-R¹ and Gal3GalNAc-R²; wherein R¹ isselected from the group consisting of ceramide or other glycolipid, R²is selected from the group consisting of Gal4GlcCer,(Neu5Ac3)Gal4GlcCer, and (Neu5Ac8Neu5c3)Gal4GlcCer. For example, forganglioside synthesis the galactosylated acceptor can be selected fromthe group consisting of Gal4GlcCer, Gal3GalNAc4(Neu5Ac3)Gal4GlcCer, andGal3GalNAc4(Neu5Ac8Neu5c3)Gal4GlcCer.

The methods and reaction mixtures of the invention are useful forproducing any of a large number of gangliosides, lysogangliosides, andrelated structures. Many gangliosides of interest are described inOettgen, H. F., ed., Gangliosides and Cancer, VCH, Germany, 1989, pp.10–15, and references cited therein. Gangliosides of particular interestinclude, for example, those found in the brain as well as other sourceswhich are listed in Table 1.

TABLE 1 Ganglioside Formulas and Abbreviations Structure AbbreviationNeu5Ac3Gal4GlcCer GM3 GalNAc4(Neu5Ac3)Gal4GlcCer GM2Gal3GalNAc4(Neu5Ac3)Gal4GlcCer GM1a Neu5Ac3Gal3GalNAc4Gal4GlcCer GM1bNeu5Ac8Neu5Ac3Gal4GlcCer GD3 GalNAc4(Neu5Ac8Neu5Ac3)Gal4GlcCer GD2Neu5Ac3Gal3GalNAc4(Neu5Ac3)Gal4GlcCer GD1aNeu5Ac3Gal3(Neu5Ac6)GalNAc4Gal4GlcCer GD1αGal3GalNAc4(Neu5Ac8Neu5Ac3)Gal4GlcCer GD1bNeu5Ac8Neu5Ac3Gal3GalNAc4(Neu5Ac3)Gal4GlcCer GT1aNeu5Ac3Gal3GalNAc4(Neu5Ac8Neu5Ac3)Gal4GlcCer GT1bGal3GalNAc4(Neu5Ac8Neu5Ac8Neu5Ac3)Gal4GlcCer GT1cNeu5Ac8Neu5Ac3Gal3GalNAc4(Neu5Ac8Neu5c3)Gal4GlcCer GQ1b Nomenclature ofGlycolipids, IUPAC-IUB Joint Commission on Biochemical Nomenclature(Recommendations 1997); Pure Appl. Chem. (1997) 69: 2475–2487; Eur. J.Biochem (1998) 257: 293–298) (www.chem.qmw.ac.uk/iupac/misc/glylp.html).

The bifunctional sialyltransferases of the invention are particularlyuseful for synthesizing the gangliosides GD1a, GD1b, GT1a, GT1b, GT1c,and GQ1b, or the carbohydrate portions of these gangliosides, forexample. The structures for these gangliosides, which are shown in Table1, requires both an α2,3- and an α2,8-sialyltransferase activity. Anadvantage provided by the methods and reaction mixtures of the inventionis that both activities are present in a single polypeptide.

The glycosyltransferases of the invention can be used in combinationwith additional glycosyltransferases and other enzymes. For example, onecan use a combination of sialyltransferase and galactosyltransferases.In some embodiments of the invention, the galactosylated acceptor thatis utilized by the bifunctional sialyltransferase is formed bycontacting a suitable acceptor with UDP-Gal and a galactosyltransferase.The galactosyltransferase polypeptide, which can be one that isdescribed herein, transfers the Gal residue from the UDP-Gal to theacceptor.

Similarly, one can use the β1,4-GalNAc transferases of the invention tosynthesize an acceptor for the galactosyltransferase. For example, theacceptor saccharide for the galactosyltransferase can formed bycontacting an acceptor for a GalNAc transferase with UDP-GalNAc and aGalNAc transferase polypeptide, wherein the GalNAc transferasepolypeptide transfers the GalNAc residue from the UDP-GalNAc to theacceptor for the GalNAc transferase.

In this group of embodiments, the enzymes and substrates can be combinedin an initial reaction mixture, or the enzymes and reagents for a secondglycosyltransferase cycle can be added to the reaction medium once thefirst glycosyltransferase cycle has neared completion. By conducting twoglycosyltransferase cycles in sequence in a single vessel, overallyields are improved over procedures in which an intermediate species isisolated. Moreover, cleanup and disposal of extra solvents andby-products is reduced.

The products produced by the above processes can be used withoutpurification. However, it is usually preferred to recover the product.Standard, well known techniques for recovery of glycosylated saccharidessuch as thin or thick layer chromatography, or ion exchangechromatography. It is preferred to use membrane filtration, morepreferably utilizing a reverse osmotic membrane, or one or more columnchromatographic techniques for the recovery.

E. Uses of Glycoconjugates Produced Using Glycosyltransferases andMethods of the Invention

The oligosaccharide compounds that are made using theglycosyltransferases and methods of the invention can be used in avariety of applications, e.g., as antigens, diagnostic reagents, or astherapeutics. Thus, the present invention also provides pharmaceuticalcompositions which can be used in treating a variety of conditions. Thepharmaceutical compositions are comprised of oligosaccharides madeaccording to the methods described above.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington 's Pharmaceutical Sciences,Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527–1533(1990).

The pharmaceutical compositions are intended for parenteral, intranasal,topical, oral or local administration, such as by aerosol ortransdermally, for prophylactic and/or therapeutic treatment. Commonly,the pharmaceutical compositions are administered parenterally, e.g.,intravenously. Thus, the invention provides compositions for parenteraladministration which comprise the compound dissolved or suspended in anacceptable carrier, preferably an aqueous carrier, e.g., water, bufferedwater, saline, PBS and the like. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9 and most preferably from 7 and 8.

In some embodiments the oligosaccharides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

The compositions containing the oligosaccharides can be administered forprophylactic and/or therapeutic treatments. In therapeutic applications,compositions are administered to a patient already suffering from adisease, as described above, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Anamount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend on theseverity of the disease and the weight and general state of the patient,but generally range from about 0.5 mg to about 40 g of oligosaccharideper day for a 70 kg patient, with dosages of from about 5 mg to about 20g of the compounds per day being more commonly used.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the oligosaccharides of this invention sufficient toeffectively treat the patient.

The oligosaccharides may also find use as diagnostic reagents. Forexample, labeled compounds can be used to locate areas of inflammationor tumor metastasis in a patient suspected of having an inflammation.For this use, the compounds can be labeled with appropriateradioisotopes, for example, ¹²⁵I, ¹⁴C, or tritium.

The oligosaccharide of the invention can be used as an immunogen for theproduction of monoclonal or polyclonal antibodies specifically reactivewith the compounds of the invention. The multitude of techniquesavailable to those skilled in the art for production and manipulation ofvarious immunoglobulin molecules can be used in the present invention.Antibodies may be produced by a variety of means well known to those ofskill in the art.

The production of non-human monoclonal antibodies, e.g., murine,lagomorpha, equine, etc., is well known and may be accomplished by, forexample, immunizing the animal with a preparation containing theoligosaccharide of the invention. Antibody-producing cells obtained fromthe immunized animals are immortalized and screened, or screened firstfor the production of the desired antibody and then immortalized. For adiscussion of general procedures of monoclonal antibody production, see,Harlow and Lane, Antibodies, A Laboratory Manual Cold Spring HarborPublications, N.Y. (1988).

EXAMPLE

The following example is offered to illustrate, but not to limit thepresent invention.

This Example describes the use of two strategies for the cloning of fourgenes responsible for the biosynthesis of the GT1 a ganglioside mimic inthe LOS of a bacterial pathogen, Campylobacter jejuni OH4384, which hasbeen associated with Guillain-Barrésyndrome (Aspinall et al. (1994)Infect. Immun. 62: 2122–2125). Aspinal et al. ((1994) Biochemistry 33:241–249) showed that this strain has an outer core LPS that mimics thetri-sialylated ganglioside GT1a. We first cloned a gene encoding anα-2,3-sialyltransferase (cst-I) using an activity screening strategy. Wethen used raw nucleotide sequence information from the recentlycompleted sequence of C. jejuni NCTC 11168 to amplify a region involvedin LOS biosynthesis from C. jejuni OH4384. Using primers that arelocated in the heptosyl-transferases I and II, the 11.47 kb LOSbiosynthesis locus from C. jejuni OH4384 was amplified. Sequencingrevealed that the locus encodes 13 partial or complete open readingframes (ORFs), while the corresponding locus in C. jejuni NCTC 11168spans 13.49 kb and contains 15 ORFs, indicating a different organizationbetween these two strains.

Potential glycosyltransferase genes were cloned individually, expressedin Escherichia coli and assayed using synthetic fluorescentoligosaccharides as acceptors. We identified genes that encode aβ-1,4-N-acetylgalactosaminyl-transferase (cgtA), aβ-1,3-galactosyltransferase (cgtB) and a bifunctional sialyltransferase(cst-II) which transfers sialic acid to O-3 of galactose and to O-8 of asialic acid that is linked α-2,3-to a galactose. The linkage specificityof each identified glycosyltransferase was confirmed by NMR analysis at600 MHz on nanomole amounts of model compounds synthesized in vitro.Using a gradient inverse broadband nano-NMR probe, sequence informationcould be obtained by detection of ³J(C, H) correlations across theglycosidic bond. The role of cgtA and cst-II in the synthesis of theGT1a mimic in C. jejuni QH4384 were confirmed by comparing theirsequence and activity with corresponding homologues in two related C.jejuni strains that express shorter ganglioside mimics in their LOS.Thus, these three enzymes can be used to synthesize a GT1a mimicstarting from lactose.

The abbreviations used are: CE, capillary electrophoresis; CMP-Neu5Ac,cytidine monophosphate-N-acetylneuraminic acid; COSY, correlatedspectroscopy; FCHASE, 6-(5-fluorescein-carboxamido)-hexanoic acidsuccimidyl ester; GBS, Guillain-Barré syndrome; HMBC, heteronuclearmultiple bond coherence; HSQC, heteronuclear single quantum coherence;LIF, laser induced fluorescence; LOS, lipooligosaccharide; LPS,lipopolysaccharide; NOE, nuclear Overhauser effect; NOESY, NOEspectroscopy; TOCSY, total correlation spectroscopy.

Experimental Procedures

Bacterial Strains

The following C. jejuni strains were used in this study: serostain O:19(ATCC #43446); serotype O:19 (strains OH4382 and OH4384 were obtainedfrom the Laboratory Centre for Disease Control (Health Canada, Winnipeg,Manitoba)); and serotype O:2 (NCTC #11168). Escherichia coli DH5α wasused for the HindE library while E. coli AD202 (CGSG #7297) was used toexpress the different cloned glycosyltransferases.

Basic Recombinant DNA Methods.

Genomic DNA isolation from the C. jejuni strains was performed usingQiagen Genomic-tip 500/G (Qiagen Inc., Valencia, Calif.) as describedpreviously (Gilbert et al. (1996) J. Biol. Chem. 271: 28271–28276).Plasmid DNA isolation, restriction enzyme digestions, purification ofDNA fragments for cloning, ligations and transformations were performedas recommended by the enzyme supplier, or the manufacturer of the kitused for the particular procedure. Long PCR reactions (>3 kb) wereperformed using the Expand™ long template PCR system as described by themanufacturer (Boehringer Mannheim, Montreal). PCR reactions to amplifyspecific ORFs were performed using the Pwo DNA polymerase as describedby the manufacturer (Boehringer Mannheim, Montreal). Restriction and DNAmodification enzymes were purchased from New England Biolabs Ltd.(Mississauga, ON). DNA sequencing was performed using an AppliedBiosystems (Montreal) model 370A automated DNA sequencer and themanufacturer's cycle sequencing kit.

Activity Screening for Sialyltransferase from C. jejuni

The genomic library was prepared using a partial HindIII digest of thechromosomal DNA of C. jejuni OH4384. The partial digest was purified ona QIAquick column (QIAGEN Inc.) and ligated with HindIII digestedpBluescript SK-. E. coli DH5α was electroporated with the ligationmixture and the cells were plated on LB medium with 150 μg/mLampicillin, 0.05 mM IPTG and 100 μg/mL X-Gal(5-Bromo4-chloro-indolyl-β-D-galactopyranoside). White colonies werepicked in pools of 100 and were resuspended in 1 mL of medium with 15%glycerol. Twenty μL of each pool were used to inoculate 1.5 mL of LBmedium supplemented with 150 μg/mL ampicillin. After 2 h of growth at37° C., IPTG was added to 1 mM and the cultures were grown for another4.5 h. The cells were recovered by centrifugation, resuspended in 0.5 mLof 50 mM Mops (pH 7, 10 mM MgCl₂) and sonicated for 1 min. The extractswere assayed for sialyltransferase activity as described below exceptthat the incubation time and temperature were 18 h and 32° C.,respectively. The positive pools were plated for single colonies, and200 colonies were picked and tested for activity in pools of 10. Finallythe colonies of the positive pools were tested individually which led tothe isolation of a two positive clones, pCJH9 (5.3 kb insert) andpCJH101 (3.9 kb insert). Using several sub-cloned fragments andcustom-made primers, the inserts of the two clones were completelysequenced on both strands. The clones with individual HindIII fragmentswere also tested for sialyltransferase activity and the insert of theonly positive one (a 1.1 kb HindIII fragment cloned in pBluescript SK-)was transferred to pUC118 using KpnI and PstI sites in order to obtainthe insert in the opposite orientation with respect to the placpromoter.

Cloning and Sequencing of the LPS Biosynthesis Locus.

The primers used to amplify the LPS biosynthesis locus of C. jejuniOH4384 were based on preliminary sequences available from the website ofthe C. jejuni sequencing group (Sanger Centre, UK) who sequenced thecomplete genome of the strain NCTC11168. The primers CJ-42 and CJ-43(all primers sequences are described in Table 2) were used to amplify an11.47 kb locus using the Expand™ long template PCR system. The PCRproduct was purified on a S-300 spin column (Pharmacia Biotech) andcompletely sequence on both strands using a combination of primerwalking and sub-cloning of HindIII fragments. Specific ORF's wereamplified using the primers described in Table 2 and the Pwo DNApolymerase. The PCR products were digested using the appropriaterestriction enzymes (see Table 2) and were cloned in pCWori+.

TABLE 2 Primers used for Amplification of Open Reading Frames Primersused to amplify the LPS core biosynthesis locus CJ42: Primer inheptosylTase-II (SEQ ID NO:40) 5′ GC CAT TAC CGT ATC GCC TAA CCA GG3′  25 mer CJ43: Primer in heptosylTase-I (SEQ ID NO:41) 5′ AAA GAA TACGAA TTT GCT AAA GAG G 3′  25 mer Primers used to amplify and clone ORF5a: CJ-106 (3′ primer, 41 mer)(SEQ ID NO:42)           SalI 5′ CCT AGGTCG ACT TAA AAC AAT GTT AAG AAT ATT TTT TTT AG 3′ CJ-157 (5′ primer, 37mer)(SEQ ID NO:43)                   NdeI 5′ CTT AGG AGG TCA TAT GCT ATTTCA ATC ATA CTT TGT G 3′ Primers used to amplify and clone ORF 6a:CJ-105 (3′ primer, 37 mer)(SEQ ID NO:44)           SalI 5′ CCT AGG TCGACC TCT AAA AAA AAT ATT CTT AAC ATT G 3′ CJ-133 (5′ primer, 39 mer)(SEQID NO:45)               NdeI 5′ CTTAGGAGGTCATATGTTTAAAATTTCAATCATCTTACC3′ Primers used to amplify and clone ORF 7a: CJ-131 (5′ primer, 41mer)(SEQ ID NO:46)               NdeI5′ CTTAGGAGGTCATATGAAAAAAGTTATTATTGCTGGAAATG 3′ CJ-132 (3′ primer, 41mer)(SEQ ID NO:47)          SalI5′ CCTAGGTCGACTTATTTTCCTTTGAAATAATGCTTTATATC 3′

Expression in E. coli and Glycosyltransferase Assays.

The various constructs were transferred to E. coli AD202 and were testedfor the expression of glycosyltransferase activities following a 4 hinduction with 1 mM IPTG. Extracts were made by sonication and theenzymatic reactions were performed overnight at 32° C. FCHASE-labeledoligosaccharides were prepared as described previously (Wakarchuk et al.(1996) J. Biol. Chem. 271: 19166–19173). Protein concentration wasdetermined using the bicinchoninic acid protein assay kit (Pierce,Rockford, Ill.). For all of the enzymatic assays one unit of activitywas defined as the amount of enzyme that generated one μmol of productper minute.

The screening assay for α-2,3-sialyltransferase activity in pools ofclones contained 1 mM Lac-FCHASE, 0.2 mM CMP-Neu5Ac, 50 mM Mops pH 7, 10mM MnCl₂ and 10 mM MgCl₂ in a final volume of 10 μL. The varioussubcloned ORFs were tested for the expression of glycosyltransferaseactivities following a 4 h induction of the cultures with 1 mM IPTG.Extracts were made by sonication and the enzymatic reactions wereperformed overnight at 32° C.

The β-1,3-galactosyltransferase was assayed using 0.2 mM GM2-FCHASE, 1mM UDP-Gal, 50 mM Mes pH 6, 10 mM MnCl₂ and 1 mM DTT. The β-1,4-GalNActransferase was assayed using 0.5 mM GM3-FCHASE, 1 mM UDP-GalNAc, 50 mMHepes pH 7 and 10 mM MnCl₂. The α-2,3-sialyltransferase was assayedusing 0.5 mM Lac-FCHASE, 0.2 mM CMP-Neu5Ac, 50 mM Hepes pH 7 and 10 mMMgCl₂. The α-2,8-sialyltransferase was assayed using 0.5 mM GM3-FCHASE,0.2 mM CMP-Neu5Ac, 50 mM Hepes pH 7 and 10 mM MnCl₂.

The reaction mixes were diluted appropriately with 10 mM NaOH andanalyzed by capillary electrophoresis performed using the separation anddetection conditions as described previously (Gilbert et al. (1996) J.Biol. Chem. 271, 28271–28276). The peaks from the electropherograms wereanalyzed using manual peak integration with the P/ACE Station software.For rapid detection of enzyme activity, samples from the transferasereaction mixtures were examined by thin layer chromatography onsilica-60 TLC plates (E. Merck) as described previously (Id.).

NMR Spectroscopy

NMR experiments were performed on a Varian INOVA 600 NMR spectrometer.Most experiments were done using a 5 mm Z gradient triple resonanceprobe. NMR samples were prepared from 0.3–0.5 mg (200–500 nanomole) ofFCHASE-glycoside. The compounds were dissolved in H₂O and the pH wasadjusted to 7.0 with dilute NaOH. After freeze drying the samples weredissolved in 600 μL D₂O. All NMR experiments were performed aspreviously described (Pavliak et al. (1993) J. Biol. Chem. 268:14146–14152; Brisson et al. (1997) Biochemistry 36: 3278–3292) usingstandard techniques such as COSY, TOCSY, NOESY, 1D-NOESY, 1D-TOCSY andHSQC. For the proton chemical shift reference, the methyl resonance ofinternal acetone was set at 2.225 ppm (¹H). For the ¹³C chemical shiftreference, the methyl resonance of internal acetone was set at 31.07 ppmrelative to external dioxane at 67.40 ppm. Homonuclear experiments wereon the order of 5–8 hours each. The 1D NOESY experiments for GD3-FCHASE,[0.3 mM], with 8000 scans and a mixing time of 800 ms was done for aduration of 8.5 h each and processed with a line broadening factor of2–5 Hz. For the 1D NOESY of the resonances at 4.16 ppm, 3000 scans wereused. The following parameters were used to acquire the HSQC spectrum:relaxation delay of 1.0 s, spectral widths in F₂ and F₁ of 6000 and24147 Hz, respectively, acquisition times in t₂ of 171 ms. For the t₁dimension, 128 complex points were acquired using 256 scans perincrement. The sign discrimination in F₁ was achieved by the Statesmethod. The total acquisition time was 20 hours. For GM2-FCHASE, due tobroad lines, the number of scans per increment was increased so that theHSQC was performed for 64 hours. The phase-sensitive spectrum wasobtained after zero filling to 2048×2048 points. Unshifted gaussianwindow functions were applied in both dimensions. The HSQC spectra wereplotted at a resolution of 23 Hz/point in the ¹³C dimension and 8Hz/point in the proton dimension. For the observation of the multipletsplittings, the ¹H dimension was reprocessed at a resolution of 2Hz/point using forward linear prediction and a π/4-shifted squaredsinebell function. All the NMR data was acquired using Varian's standardsequences provided with the VNMR 5.1 or VNMR 6.1 software. The sameprogram was used for processing.

A gradient inverse broadband nano-NMR probe (Varian) was used to performthe gradient HMBC (Bax and Summers (1986) J. Am. Chem. Soc. 108,2093–2094; Parella et al. (1995) J. Mag. Reson. A 112, 241–245)experiment for the GD3-FCHASE sample. The nano-NMR probe which is ahigh-resolution magic angle spinning probe produces high resolutionspectra of liquid samples dissolved in only 40 μL (Manzi et al. (1995)J. Biol. Chem. 270, 9154–9163). The GD3-FCHASE sample (mass=1486.33 Da)was prepared by lyophilizing the original 0.6 mL sample (200 nanomoles)and dissolving it in 40 μL of D₂O for a final concentration of 5 mM. Thefinal pH of the sample could not be measured.

The gradient HMBC experiment was done at a spin rate of 2990 Hz, 400increments of 1024 complex points, 128 scans per increment, acquisitiontime of 0.21 s, ¹J(C, H)=140 Hz and ^(n)J(C, H)=8 Hz, for a duration of18.5 h.

Mass Spectrometry

All mass measurements were obtained using a Perkin-Elmer Biosystems(Framingham, Mass.) Elite-STR MALDI-TOF instrument. Approximately two μgof each oligosaccharide was mixed with a matrix containing a saturatedsolution of dihydroxybenzoic acid. Positive and negative mass spectrawere acquired using the reflector mode.

Results

Detection of Glycosyltransferase Activities in C. jejuni Strains

Before the cloning of the glycosyltransferase genes, we examined C.jejuni OH4384 and NCTC 11168 cells for various enzymatic activities.When an enzyme activity was detected, the assay conditions wereoptimized (described in the Experimental Procedures) to ensure maximalactivity. The capillary electrophoresis assay we employed was extremelysensitive and allowed detection of enzyme activity in the μU/ml range(Gilbert et al. (1996) J. Biol. Chem. 271: 28271–28276). We examinedboth the sequenced strain NCTC 11168 and the GBS-associated strainOH4384 for the enzymes required for the GT1a ganglioside mimicsynthesis. As predicted, strain OH4384 possessed the enzyme activitiesrequired for the synthesis of this structure:β-1,4-N-acetylgalactosaminyltransferase, β-1,3-galactosyltransferase,β-2,3-sialyltransferase and α-2,8-sialyltransferase. The genome of thestrain, NCTC 11168 lacked the β-1,3-galactosyltransferase and theα-2,8-sialyltransferase activities.

Cloning of an α-2,3-sialyltransferase (cst-I) Using an ActivityScreening Strategy

A plasmid library made from an unfractionated partial HindIII digestionof chromosomal DNA from C. jejuni OH4384 yielded 2,600 white colonieswhich were picked to form pools of 100. We used a “divide and conquer”screening protocol from which two positive clones were obtained anddesignated pCJH9 (5.3 kb insert, 3 HindIII sites) and pCJH101 (3.9 kbinsert, 4 HindIII sites). Open reading frame (ORF) analysis and PCRreactions with C. jejuni OH4384 chromosomal DNA indicated that pCJH9contained inserts that were not contiguous in the chromosomal DNA. Thesequence downstream of nucleotide #1440 in pCJH9 was not further studiedwhile the first 1439 nucleotides were found to be completely containedwithin the sequence of pCJH101. The ORF analysis and PCR reactions withchromosomal DNA indicated that all of the pCJH101 HindIII fragments werecontiguous in C. jejuni OH4384 chromosomal DNA.

Four ORFs, two partial and two complete, were found in the sequence ofpCJH101 (FIG. 2). The first 812 nucleotides encode a polypeptide that is69% identical with the last 265 a.a. residues of the peptide chainrelease factor RF-2 (prfB gene, GenBank #AE000537) from Helicobacterpylori. The last base of the TAA stop codon of the chain release factoris also the first base of the ATG start codon of an open reading framethat spans nucleotides #812 to #2104 in pCJH101. This ORF was designatedcst-I (Campylobacter sialyltransferase I) and encodes a 430 amino acidpolypeptide that is homologous with a putative ORF from Haemophilusinfluenzae (GenBank #U32720). The putative H. influenzae ORF encodes a231 amino acid polypeptide that is 39% identical to the middle region ofthe Cst I polypeptide (amino acid residues #80 to #330). The sequencedownstream of cst-I includes an ORF and a partial ORF that encodepolypeptides that are homologous (>60% identical) with the two subunits,CysD and CysN, of the E. coli sulfate adenylyltransferase (GenBank#AE000358).

In order to confirm that the cst-I ORE encodes sialyltransferaseactivity, we sub-cloned it and over-expressed it in E. coli. Theexpressed enzyme was used to add sialic acid to Gal-β-1,4-Glc-β-FCHASE(Lac-FCHASE). This product (GM3-FCHASE) was analyzed by NMR to confirmthe Neu5Ac-α-2,3-Gal linkage specificity of Cst-I.

Sequencing of the LOS Biosynthesis Locus of C. jejuni OH4384

Analysis of the preliminary sequence data available at the website ofthe C. jejuni NCTC 11168 sequencing group (Sanger Centre, UK) revealedthat the two heptosyltransferases involved in the synthesis of the innercore of the LPS were readily identifiable by sequence homology withother bacterial heptosyltransferases. The region between the twoheptosyltransferases spans 13.49 kb in NCTC 11168 and includes at leastseven potential glycosyltransferases based on BLAST searches in GenBank.Since no structure is available for the LOS outer core of NCTC 11168, itwas impossible to suggest functions for the putative glycosyltransferasegenes in that strain.

Based on conserved regions in the heptosyltransferases sequences, wedesigned primers (CJ-42 and CJ-43) to amplify the region between them.We obtained a PCR product of 13.49 kb using chromosomal DNA from C.jejuni NCTC 11168 and a PCR product of 11.47 kb using chromosomal DNAfrom C. jejuni OH4384. The size of the PCR product from strain NCTC11168 was consistent with the Sanger Centre data. The smaller size ofthe PCR product from strain OH4384 indicated heterogeneity between thestrains in the region between the two heptosyltransferase genes andsuggested that the genes for some of the glycosyltransferases specificto strain OH4384 could be present in that location. We sequenced the11.47 kb PCR product using a combination of primer walking andsub-cloning of HindIII fragments (GenBank #AF130984). The G/C content ofthe DNA was 27%, typical of DNA from Campylobacter. Analysis of thesequence revealed eleven complete ORFs in addition to the two partialORFs encoding the two heptosyltransferases (FIG. 2, Table 3). Whencomparing the deduced amino acid sequences, we found that the twostrains share six genes that are above 80% identical and four genes thatare between 52 and 68% identical (Table 3). Four genes are unique to C.jejuni NCTC 11168 while one gene is unique to C. jejuni OH4384 (FIG. 2).Two genes that are present as separate ORFs (ORF #5a and #10a) in C.jejuni OH4384 are found in an in-frame fusion ORF (#5b/10b) in C. jejuniNCTC 11168.

TABLE 3 Location and description of the ORFs of the LOS biosynthesislocus from C. jejuni OH4384 Homologue in Strain NCTC11168^(a) (%identity Homologues found in in the GenBank a.a. (% identity in the a.aORF # Location sequence) sequence) Function^(b)  1a    1–357 ORF #1brfaC (GB #AE000546) Heptosyltransferase I (98%) from Helicobacter pylori(35%)  2a   350–1,234 ORF #2b waaM (GB Lipid A biosynthesis (96%)#AE001463) from acyltransferase Helicobacter pylori (25%)  3a 1,234–2,487 ORF #3b lgtF (GB #U58765) Glycosyltransferase (90%) fromNeisseria meningitidis (31%)  4a  2,786–3,952 ORF #4b cps14J (GB#X85787) Glycosyltransferase (80%) from Streptococcus pneumoniae (45%over first 100 a.a)  5a  4,025–5,065 N-terminus of ORF #HP0217 (GBβ-1,4-N-acetylgalac- ORF #5b/10b #AE000541) tosaminyltransferase (52%)from Helicobacter (cgtA) pylori (50%)  6a  5,057–5,959 ORF #6b cps23FU(GB β-1,3-Galactosyltransferase (complement) (60%) #AF030373) from(cgtB) Streptococcus pneumoniae (23%)  7a  6,048–6,920 ORF #7b ORF#HI0352 (GB Bi-functional α- (52%) #U32720) from 2,3/α2,8sialyltransferase Haemophilus (cst-II) influenzae (40%)  8a  6,924–7,961ORF #8b siaC (GB #U40740) Sialic acid synthase (80%) from Neisseriameningitidis (56%)  9a  8,021–9,076 ORF #9b siaA (GB #M95053) Sialicacid biosynthesis (80%) from Neisseria meningitidis (40%) 10a 9,076–9,738 C-terminus of neuA (GB #U54496) CMP-sialic acid ORF #5b/10bfrom synthetase (68%) Haemophilus ducreyi (39%) 11a  9,729–10,559 NoPutative ORF (GB Acetyltransferase homologue #AF010496) from Rhodobactercapsulatus (22%) 12a 10,557–11,366 ORF #12b ORF #HI0868 (GBGlycosyltransferase (complement) (90%) #U32768) from Haemophilusinfluenzae (23%) 13a 11,347–11,474 ORF #13b rfaF (GB #AE000625)Heptosyltransferase II (100%) from Helicobacter pylori (60%) ^(a)Thesequence of the C. jejuni NCTC 11168 ORFs can be obtained from theSanger Centre. ^(b)The functions that were determined experimentally arein bold fonts. Other functions are based on higher score homologues fromGenBank.

Identification of Outer Core Glycosyltransferases

Various constructs were made to express each of the potentialglycosyltransferase genes located between the two heptosyltransferasesfrom C. jejuni OH4384. The plasmid pCJL-09 contained the ORF #5a and aculture of this construct showed GalNAc transferase activity whenassayed using GM3-FCHASE as acceptor. The GalNAc transferase wasspecific for a sialylated acceptor since Lac-FCHASE was a poor substrate(less than 2% of the activity observed with GM3-FCHASE). The reactionproduct obtained from GM3-FCHASE had the correct mass as determined byMALDI-TOF mass spectrometry, and the identical elution time in the CEassay as the GM2-FCHASE standard. Considering the structure of the outercore LPS of C. jejuni OH4384, this GalNAc transferase (cgtA forCamplyobacter glycosyltransferase A), has a β-1,4-specificity to theterminal Gal residue of GM3-FCHASE. The linkage specificity of CgtA wasconfirmed by the NMR analysis of GM2-FCHASE (see text below, Table 4).The in vivo role of cgtA in the synthesis of a GM2 mimic is confirmed bythe natural knock-out mutant provided by C. jejuni OH4382 (FIG. 1). Uponsequencing of the cgtA homologue from C. jejuni OH4382 we found aframe-shift mutation (a stretch of seven A's instead of 8 A's after base#71) which would result in the expression of a truncated cgtA version(29 aa instead of 347 aa). The LOS outer core structure of C. jejuniOH4382 is consistent with the absence of β-1,4-GlaNAc transferase as theinner galactose residue is substituted with sialic acid only (Aspinallet al. (1994) Biochemistry 33, 241–249).

The plasmid pCJL-04 contained the ORF #6a and an IPTG-induced culture ofthis construct showed galactosyltransferase activity using GM2-FCHASE asan acceptor thereby producing GM1a-FCHASE. This product was sensitive toβ-1,3-galactosidase and was found to have the correct mass by MALDI-TOFmass spectrometry. Considering the structure of the LOS outer core of C.jejuni OH4384, we suggest that this galactosyltransferase (cgtB forCampylobacter glycosyltransferase B) has β-1,3-specificity to theterminal GalNAc residue of GM2-FCHASE. The linkage specificity of CgtAwas confirmed by the NMR analysis of GM1a-FCHASE (see text below, Table4) which was synthesized by using sequentially Cst-I, CgtA and CgtB.

The plasmid pCJL-03 included the ORF #7a and an IPTG-induced cultureshowed sialyltransferase activity using both Lac-FCHASE and GM3-FCHASEas acceptors. This second sialyltransferase from OH4384 was designatedcst-II. Cst-II was shown to be bi-functional as it could transfer sialicacid α-2,3 to the terminal Gal of Lac-FCHASE and also α-2,8-to theterminal sialic acid of GM3-FCHASE. NMR analysis of a reaction productformed with Lac-FCHASE confirmed the α-2,3-linkage of the first sialicacid on the Gal, and the α-2,8-linkage of the second sialic acid (seetext below, Table 4).

TABLE 4 Proton NMR chemical shifts^(a) for the fluorescent derivativesof the ganglioside mimics synthesized using the clonedglycosyltransferases. Chemical Shift (ppm) Residue H Lac- GM3- GM2-GM1a- GD3- βGlc 1 4.57 4.70 4.73 4.76 4.76 a 2 3.23 3.32 3.27 3.30 3.383 3.47 3.54 3.56 3.58 3.57 4 3.37 3.48 3.39 3.43 3.56 5 3.30 3.44 3.443.46 3.50 6 3.73 3.81 3.80 3.81 3.85 6′ 3.22 3.38 3.26 3.35 3.50βGal(1-4) 1 4.32 4.43 4.42 4.44 4.46 b 2 3.59 3.60 3.39 3.39 3.60 3 3.694.13 4.18 4.18 4.10 4 3.97 3.99 4.17 4.17 4.00 5 3.81 3.77 3.84 3.833.78 6 3.86 3.81 3.79 3.78 3.78 6′ 3.81 3.78 3.79 3.78 3.78 αNeu5Ac(2-3)3_(ax) 1.81 1.97 1.96 1.78 c 3_(eq) 2.76 2.67 2.68 2.67 4 3.69 3.78 3.793.60 5 3.86 3.84 3.83 3.82 6 3.65 3.49 3.51 3.68 7 3.59 3.61 3.60 3.87 83.91 3.77 3.77 4.15 9 3.88 3.90 3.89 4.18 9′ 3.65 3.63 3.64 3.74 NAc2.03 2.04 2.03 2.07 βGalNAc(1-4) 1 4.77 4.81 d 2 3.94 4.07 3 3.70 3.82 43.93 4.18 5 3.74 3.75 6 3.86 3.84 6′ 3.86 3.84 NAc 2.04 2.04 βGal(1-3) 14.55 e 2 3.53 3 3.64 4 3.92 5 3.69 6 3.78 6′ 3.74 αNeu5Ac(2-8) 3_(ax)1.75 f 3_(eq) 2.76 4 3.66 5 3.82 6 3.61 7 3.58 8 3.91 9 3.88 9′ 3.64 NAc2.02 ^(a)in ppm from HSQC spectrum obtained at 600 MHz, D₂O, pH 7, 28°C. for Lac-, 25° C. for GM3-, 16° C. for GM2-, 24° C. for GM1a-, and 24°C. GD3-FCHASE. The methyl resonance of internal acetone is at 2.225 ppm(¹H). The error is ±0.02 ppm for ¹H chemical shifts and ±5° C. for thesample temperature. The error is ±0.1 ppm for the H-6 resonances ofresidue a, b, d and e due to overlap.

Comparison of the Sialyltransferases

The in vivo role of cst-II from C. jejuni OH4384 in the synthesis of atri-sialylated GT1a ganglioside mimic is supported by comparison withthe cst-II homologue from C. jejuni O:19 (serostrain) that expresses thedi-sialylated GD1a ganglioside mimic. There are 24 nucleotidedifferences that translate into 8 amino acid differences between thesetwo cst-II homologues (FIG. 3). When expressed in E. coli, the cst-IIhomologue from C. jejuni O:19 (serostrain) has α-2,3-sialyltransferaseactivity but very low α-2,8-sialyltransferase activity (Table 5) whichis consistent with the absence of terminal α-2,8-linked sialic acid inthe LOS outer core (Aspinall et al. (1994) Biochemistry 33, 241–249) ofC. jejuni O:19 (serostrain). The cst-II homologue from C. jejuni NCTC11168 expressed much lower α-2,3-sialyltransferase activity than thehomologues from O:19 (serostrain) or OH4384 and no detectableα-2,8-sialyltransferase activity. We could detect an IPTG-inducible bandon a SDS-PAGE gel when cst-II from NCTC 11168 was expressed in E. coli(data not shown). The Cst-II protein from NCTC 11168 shares only 52%identity with the homologues from O:19 (serostrain) or OH4384. We couldnot determine whether the sequence differences could be responsible forthe lower activity expressed in E. coli.

Although cst-I mapped outside the LOS biosynthesis locus, it isobviously homologous to cst-II since its first 300 residues share 44%identity with Cst-II from either C. jejuni OH4384 or C. jejuni NCTC11168 (FIG. 3). The two Cst-II homologues share 52% identical residuesbetween themselves and are missing the C-terminal 130 amino acids ofCst-I. A truncated version of Cst-I which was missing 102 amino acids atthe C-terminus was found to be active (data not shown) which indicatesthat the C-terminal domain of Cst-I is not necessary forsialyltransferase activity. Although the 102 residues at the C-terminusare dispensable for in vitro enzymatic activity, they may interact withother cell components in vivo either for regulatory purposes or forproper cell localization. The low level of conservation between the C.jejuni sialyltransferases is very different from what was previouslyobserved for the α-2,3-sialyltransferases from N. meningitidis and N.gonorrhoeae, where the 1st transferases are more than 90% identical atthe protein level between the two species and between different isolatesof the same species (Gilbert et al., supra.).

TABLE 5 Comparison of the activity of the sialyltransferases from C.jejuni. The various sialyltransferases were expressed in E. coli asfusion proteins with the maltose-binding protein in the vector pCWori+(Wakarchuk et al. (1994) Protein. Sci. 3, 467–475). Sonicated extractswere assayed using 500 μM of either Lac-FCHASE or GM3-FCHASE.Sialyltransferase Activity (μU/mg)^(a) gene Lac-FCHASE GM3-FCHASE Ratio(%)^(b) cst-I (OH4384) 3,744 2.2 0.1 cst-II (OH4384)   209 350.0 167.0cst-II (O:19 serostrain) 2,084 1.5 0.1 cst-II (NCTC 11168)    8 0 0.0^(a)The activity is expressed in μU (pmol of product per minute) per mgof total protein in the extract. ^(b)Ratio (in percentage) of theactivity on GM3-FCHASE divided by the activity on Lac-FCHASE.

NMR Analysis on Nanomole Amounts of the Synthesized Model Compounds.

In order to properly assess the linkage specificity of an identifiedglycosyltransferase, its product was analyzed by NMR spectroscopy. Inorder to reduce the time needed for the purification of the enzymaticproducts, NMR analysis was conducted on nanomole amounts. All compoundsare soluble and give sharp resonances with linewidths of a few Hz sincethe H-1 anomeric doublets (J_(1,2)=8 Hz) are well resolved. The onlyexception is for GM2-FCHASE which has broad lines (˜10 Hz), probably dueto aggregation. For the proton spectrum of the 5 mM GD3-FCHASE solutionin the nano-NMR probe, the linewidths of the anomeric signals were onthe order of 4 Hz, due to the increased concentration. Also, additionalpeaks were observed, probably due to degradation of the sample withtime. There were also some slight chemical shifts changes, probably dueto a change in pH upon concentrating the sample from 0.3 mM to 5 mM.Proton spectra were acquired at various temperatures in order to avoidoverlap of the HDO resonance with the anomeric resonances. As can beassessed from the proton spectra, all compounds were pure and impuritiesor degradation products that were present did not interfere with the NMRanalysis which was performed as previously described (Pavliak et al.(1993) J. Biol. Chem. 268, 14146–14152; Brisson et al. (1997)Biochemistry 36, 3278–3292).

For all of FCHASE glycosides, the ¹³C assignments of similar glycosides(Sabesan and Paulson (1986) J. Am. Chem. Soc. 108, 2068–2080; Michon etal. (1987) Biochemistry 26, 8399–8405; Sabesan et al. (1984) Can. J.Chem. 62, 1034–1045) were available. For the FCHASE glycosides, the ¹³Cassignments were verified by first assigning the proton spectrum fromstandard homonuclear 2D experiments, COSY, TOCSY and NOESY, and thenverifying the ¹³C assignments from an HSQC experiment, which detects C—Hcorrelations. The HSQC experiment does not detect quaternary carbonslike C-1 and C-2 of sialic acid, but the HMBC experiment does. Mainlyfor the Glc resonances, the proton chemical shifts obtained from theHSQC spectra differed from those obtained from homonuclear experimentsdue to heating of the sample during ¹³C decoupling. From a series ofproton spectrum acquired at different temperatures, the chemical shiftsof the Glc residue were found to be the most sensitive to temperature.In all compounds, the H-1 and H-2 resonances of Glc changed by 0.004ppm/° C., the Gal(1-4) H-1 by 0.002 ppm/° C., and less than 0.001 ppm/°C. for the Neu5Ac H-3 and other anomeric resonances. For LAC-FCHASE, theGlc H-6 resonance changed by 0.008 ppm/° C.

The large temperature coefficient for the Glc resonances is attributedto ring current shifts induced by the linkage to the aminophenyl groupof FCHASE. The temperature of the sample during the HSQC experiment wasmeasured from the chemical shift of the Glc H-1 and H-2 resonances. ForGM1a-FCHASE, the temperature changed from 12° C. to 24° C. due to thepresence of the Na+ counterion in the solution and NaOH used to adjustthe pH. Other samples had less severe heating (<5° C.). In all cases,changes of proton chemical shifts with temperature did not cause anyproblems in the assignments of the resonances in the HSQC spectrum. InTable 4 and Table 6, all the chemical shifts are taken from the HSQCspectra.

The linkage site on the aglycon was determined mainly from a comparisonof the ¹³C chemical shifts of the enzymatic product with those of theprecursor to determine glycosidation shifts as done previously for tensialyloligosaccharides (Salloway et al. (1996) Infect. Immun. 64,2945–2949). Here, instead of comparing ¹³C spectra, HSQC spectra arecompared, since one hundred times more material would be needed toobtain a ¹³C spectrum. When the ¹³C chemical shifts from HSQC spectra ofthe precursor compound are compared to those of the enzymatic product,the main downfield shift always occurs at the linkage site while otherchemical shifts of the precursor do not change substantially. Protonchemical shift differences are much more susceptible to long-rangeconformational effects, sample preparation, and temperature. Theidentity of the new sugar added can quickly be identified from acomparison of its ¹³C chemical shifts with those of monosaccharides orany terminal residue, since only the anomeric chemical shift of theglycon changes substantially upon glycosidation (Sabesan and Paulson,supra.).

Vicinal proton spin-spin coupling (J_(HH)) obtained from 1D TOCSY or 1DNOESY experiments also are used to determine the identity of the sugar.NOE experiments are done to sequence the sugars by the observation ofNOEs between the anomeric glycon protons (H-3s for sialic acid) and theaglycon proton resonances. The largest NOE is usually on the linkageproton but other NOEs can also occur on aglycon proton resonances thatare next to the linkage site. Although at 600 MHz, the NOEs of manytetra- and pentasaccharides are positive or very small, all thesecompounds gave good negative NOEs with a mixing time of 800 ms, probablydue to the presence of the large FCHASE moiety.

For the synthetic Lac-FCHASE, the ¹³C assignments for the lactose moietyof Lac-FCHASE were confirmed by the 2D methods outlined above. All theproton resonances of the Glc unit were assigned from a 1D-TOCSYexperiment on the H-1 resonance of Glc with a mixing time of 180 ms. A1D-TOCSY experiment for Gal H-1 was used to assign the H-1 to H-4resonances of the Gal unit. The remaining H-5 and H-6s of the Gal unitwere then assigned from the HSQC experiment. Vicinal spin-spin couplingvalues (J_(HH)) for the sugar units were in accord with previous data(Michon et al., supra.). The chemical shifts for the FCHASE moiety havebeen given previously (Gilbert et al. (1996) J. Biol. Chem. 271,28271–28276).

Accurate mass determination of the enzymatic product of Cst-I fromLac-FCHASE was consistent with the addition of sialic acid to theLac-FCHASE acceptor (FIG. 4). The product was identified as GM3-FCHASEsince the proton spectrum and ¹³C chemical shifts of the sugar moiety ofthe product (Table 6) were very similar to those for the GM3oligosaccharide or sialyllactose, (αNeu5Ac(2-3)βGal(1-4)pGlc; Sabesanand Paulson, supra.). The proton resonances of GM3-FCHASE were assignedfrom the COSY spectrum, the HSQC spectrum, and comparison of the protonand ¹³C chemical shifts with those ofαNeu5Ac(2-3)βGal(1-4)βGlcNAc-FCHASE (Gilbert et al., supra.). For thesetwo compounds, the proton and ¹³C chemical shifts for the Neu5Ac and Galresidues were within error bounds of each other (Id.). From a comparisonof the HSQC spectra of Lac-FCHASE and GM3-FCHASE, it is obvious that thelinkage site is at Gal C-3 due to the large downfield shift for Gal H-3and Gal C-3 upon sialylation typical for (2-3) sialyloligosaccarides(Sabesan and Paulson, supra.). Also, as seen before forαNeu5Ac(2-3)βGal(1-4)βGlcNAc-FCHASE (Gilbert et al., supra.), the NOEfrom H-3_(ax) of sialic acid to H-3 of Gal was observed typical of theαNeu5Ac(2-3)Gal linkage.

TABLE 6 Comparison of the ¹³C chemical shifts for the FCHASEglycosides^(a) with those observed for lactose^(b) (Sabesan and Paulson,supra.), ganglioside oligosaccharides^(b) (Id., Sabesan et al. (1984)Can. J. Chem. 62, 1034–1045) and (-8NeuAc2-)₃ (Michon et al. (1987)Biochemistry 26, 8399–8405). The chemical shifts at the glycosidationsites are underlined. Chemical Shift (ppm) Residue C Lac- Lactose GM3-GM3OS GM2- GM2OS GM1a- GM1aOS GD3- 8NeuAc2 βGlc 1 100.3  96.7 100.3 96.8 100.1  96.6 100.4  96.6 100.6  a 2 73.5 74.8 73.4 74.9 73.3 74.673.3 74.6 73.5 3 75.2 75.3 75.0 75.4 75.3 75.2 75.0 75.2 75.0 4 79.479.4 79.0 79.4 79.5 79.5 79.5 79.5 78.8 5 75.9 75.7 75.7 75.8 75.8 75.675.7 75.6 75.8 6 61.1 61.1 60.8 61.2 61.0 61.0 60.6 61.0 60.8 βGal(1-4)1 104.1  103.8  103.6  103.7  103.6  103.5  103.6  103.5  103.6  b 272.0 71.9 70.3 70.4 71.0 70.9 70.9 70.9 70.3 3 73.5 73.5 76.4 76.6 75.375.6^(c) 75.1 75.2^(c) 76.3 4 69.7 69.5 68.4 68.5 78.3 78.0^(c) 78.178.0^(c) 68.5 5 76.4 76.3 76.0 76.2 75.0 74.9 74.9 75.0 76.1 6 62.1 62.062.1 62.0 62.2 61.4 62.0 61.5 62.0 αNeu5Ac 3 40.4 40.7 37.7 37.9 37.837.9 40.4 41.7 (2-3) 4 69.2 69.3 69.8 69.5 69.5 69.5 69.0  68.8^(d) c 552.6 52.7 52.7 52.5 52.6 52.5 53.0 53.2 6 73.7 73.9 74.0 73.9 73.8 73.974.9  74.5^(d) 7 69.0 69.2 69.0 68.8 69.0 68.9 70.3 70.0 8 72.6 72.873.3 73.1 73.1 73.1 79.1 79.1 9 63.4 63.7 63.9 63.7 63.7 63.7 62.5 62.1NAc 22.9 23.1 23.2 22.9 23.3 22.9 23.2 23.2 βGalNAc 1 103.8  103.6 103.4  103.4  (1-4) 2 53.2 53.2 52.0 52.0 d 3 72.3 72.2 81.4 81.2 4 68.868.7 68.9 68.8 5 75.6 75.2 75.1 75.2 6 61.8 62.0 61.5 62.0 NAc 23.2 23.523.4 23.5 βGal(1-3) 1 105.5  105.6  e 2 71.5 71.6 3 73.1 73.4 4 69.569.5 5 75.7 75.8 6 61.9 61.8 αNeu5Ac 3 41.2 41.2 (2-8) 4 69.5 69.3 f 553.0 52.6 6 73.6 73.5 7 69.0 69.0 8 72.7 72.6 9 63.5 63.4 NAc 23.0 23.1^(a)in ppm from the HSQC spectrum obtained at 600 MHz, D₂O, pH 7, 28° C.for Lac-, 25° C. for GM3-, 16° C. for GM2-, 24° C. for GM1a-, and 24° C.GD3-FCHASE. The methyl resonance of internal acetone is at 31.07 ppmrelative to external dioxane at 67.40 ppm. The error is ±0.2 ppm for ¹³Cchemical shifts and ±5° C. for the sample temperature. The error is ±0.8ppm for 6a, 6b, 6d, 6e due to overlap. ^(b)A correction of +0.52 ppm wasadded to the chemical shifts of the reference compounds (25, 27) to makethem relative to dioxane set at 67.40 ppm. Differences of over 1 ppmbetween the chemical shifts of the FCHASE compound and the correspondingreference compound are indicated in bold. ^(c)C-3 and C-4 assignmentshave been reversed. ^(d)C-4 and C-6 assignments have been reversed.

Accurate mass determination of the enzymatic product of Cst-II fromLac-FCHASE indicated that two sialic acids had been added to theLac-FCHASE acceptor (FIG. 4). The proton resonances were assigned fromCOSY, 1D TOCSY and 1D NOESY and comparison of chemical shifts with knownstructures. The Glc H-1 to H-6 and Gal H-1 to H-4 resonances wereassigned from 1D TOCSY on the H-1 resonances. The Neu5Ac resonances wereassigned from COSY and confirmed by 1D NOESY. The 1D NOESY of the H-8,H-9-Neu5Ac resonances at 4.16 ppm was used to locate the H-9s and H-7resonances (Michon et al, supra.). The singlet appearance of the H-7resonance of Neu5Ac(2-3) arising from small vicinal coupling constantsis typical of the 2-8 linkage (Id.). The other resonances were assignedfrom the HSQC spectrum and ¹³C assignments for terminal sialic acid(Id.). The proton and ¹³C carbon chemical shifts of the Gal unit weresimilar to those in GM3-FCHASE, indicating the presence of theαNeu5Ac(2-3)Gal linkage. The J_(HH) values, proton and ¹³C chemicalshifts of the two sialic acids were similar to those ofαNeu5Ac(2-8)Neu5Ac in the α(2-8)-linked Neu5Ac trisaccharide (Sallowayet al. (1996) Infect. Immun. 64, 2945–2949) indicating the presence ofthat linkage. Hence, the product was identified as GD3-FCHASE.Sialylation at C-8 of Neu5Ac caused a downfield shift of −6.5 ppm in itsC-8 resonance from 72.6 ppm to 79.1 ppm.

The inter-residue NOEs for GD3-FCHASE were also typical of theαNeu5Ac(2-8)αNeu5Ac(2-3)βGal sequence. The largest inter-residue NOEsfrom the two H-3_(ax) resonances at 1.7–1.8 ppm of Neu5Ac(2-3) andNeu5Ac(2-8) are to the Gal H-3 and -8)Neu5Ac H-8 resonances. Smallerinter-residue NOEs to Gal H-4 and -8)Neu5Ac H-7 are also observed. NOEson FCHASE resonances are also observed due the overlap of an FCHASEresonance with the H-3_(ax) resonances (Gilbert et al., supra.). Theinter-residue NOE from H-³ _(eq) of Neu5Ac(2-3) to Gal H-3 is alsoobserved. Also, the intra-residues confirmed the proton assignments. TheNOEs for the 2-8 linkage are the same as those observed for the-8Neu5Acα2-polysaccharide (Michon et al., supra.).

The sialic acid glycosidic linkages could also be confirmed by the useof the HMBC experiment which detects ³J(C, H) correlations across theglycosidic bond. The results for both α-2,3 and α-2,8 linkages indicatethe ³J(C, H) correlations between the two Neu5Ac anomeric C-2 resonancesand Gal H-3 and -8)Neu5Ac H-8 resonances. The intra-residue correlationsto the H-3_(ax) and H-³ _(eq) resonances of the two Neu5Ac residues werealso observed. The Glc (C-1, H-2) correlation is also observed sincethere was partial overlap of the crosspeaks at 101 ppm with thecrosspeaks at 100.6 ppm in the HMBC spectrum.

Accurate mass determination of the enzymatic product of CgtA fromGM3-FCHASE indicated that a N-acetylated hexose unit had been added tothe GM3-FCHASE acceptor (FIG. 4). The product was identified asGM2-FCHASE since the glycoside proton and ¹³C chemical shifts weresimilar to those for GM2 oligosaccharide (GM2OS) (Sabesan et al. (1984)Can. J. Chem. 62, 1034–1045). From the HSQC spectrum for GM2-FCHASE andthe integration of its proton spectrum, there are now two resonances at4.17 ppm and 4.18 ppm along with a new anomeric “d1” and two NAc groupsat 2.04 ppm. From TOCSY and NOESY experiments, the resonance at 4.18 ppmwas unambiguously assigned to Gal H-3 because of the strong NOE betweenH-1 and H-3. For βgalactopyranose, strong intra-residue NOEs between H-1and H-3 and H-1 and H-5 are observed due to the axial position of theprotons and their short interproton distances (Pavliak et al. (1993) J.Biol. Chem. 268, 14146–14152; Brisson et al. (1997) Biochemistry 36,3278–3292; Sabesan et al. (1984) Can. J. Chem. 62, 1034–1045). From theTOCSY spectrum and comparison of the H1 chemical shifts of GM2-FCHASEand GM2OS (Sabesan et al., supra.) the resonance at 4.17 ppm is assignedas Gal H-4. Similarly, from TOCSY and NOESY spectra, the H-1 to H-5 ofGalNAc and Glc, and H-3 to H-6 of Neu5Ac were assigned. Due to broadlines, the multiplet pattern of the resonances could not be observed.The other resonances were assigned from comparison with the HSQCspectrum of the precursor and ¹³C assignments for GM2OS (Sabesan et al.,supra.). By comparing the HSQC spectra for GM3- and GM2-FCHASEglycosides, a −9.9 ppm downfield shift between the precursor and theproduct occurred on the Gal C-4 resonance. Along with intra-residue NOEsto H-3 and H-5 of GalNAc, the inter-residue NOE from GalNAc H-1 to GalH-4 at 4.17 ppm was also observed confirming the βGalNAc(1-4)Galsequence. The observed NOEs were those expected from the conformationalproperties of the GM2 ganglioside (Sabesan et al., supra.).

Accurate mass determination of the enzymatic product of CgtB fromGM2-FCHASE indicated that a hexose unit had been added to the GM2-FCHASEacceptor (FIG. 4). The product was identified as GM1a-FCHASE since theglycoside ¹³C chemical shifts were similar to those for the GM1aoligosaccharide (Id.). The proton resonances were assigned from COSY, 1DTOCSY and 1D NOESY. From a 1D TOCSY on the additional “e1” resonance ofthe product, four resonances with a mutltiplet pattern typical ofβ-galactopyranose were observed. From a 1D TOCSY and 1D NOESY on the H-1resonances of βGalNAc, the H-1 to H-5 resonances were assigned. TheβGalNAc H-1 to H-4 multiplet pattern was typical of theβ-galactopyranosyl configuration, confirming the identity of this sugarfor GM2-FCHASE. It was clear that upon glylcosidation, the majorperturbations occurred for the βGalNAc resonances, and there was −9.1ppm downfield shift between the acceptor and the product on the GalNAcC-3 resonance. Also, along with intra-residue NOEs to H-3, H-5 of Gal,an inter-residue NOE from Gal H-1 to GalNAc H-3 and a smaller one toGalNAc H-4 were observed, confirming the βGal(1-3)GalNAc sequence. Theobserved NOEs were those expected from the conformational properties ofthe GM1a ganglioside (Sabesan et al., supra.).

There was some discrepancy with the assignment of the C-3 and C-4βGal(1-4) resonances in GM2OS and GM1OS which are reversed from thepublished data (Sabesan et al., supra.). Previously, the assignmentswere based on comparison of ¹³C chemical shifts with known compounds.For GM1a-FCHASE, the assignment for H-3 of Gal(1-4) was confirmed byobserving its large vicinal coupling, J_(2,3)=10 Hz, directly in theHSQC spectrum processed with 2 Hz/point in the proton dimension. The H-4multiplet is much narrower (<5 Hz) due to the equatorial position of H-4in galactose (Sabesan et al., supra.). In Table 6, the C-4 and C-6assignments of one of the sialic acids in (-8Neu5Ac2-)₃ also had to bereversed (Michon et al., supra.) as confirmed from the assignments ofH-4 and H-6.

The ¹³C chemical shifts of the FCHASE glycosides obtained from HSQCspectra were in excellent agreement with those of the referenceoligosaccharides shown in Table 6. Differences of over 1 ppm wereobserved for some resonances and these are due to different aglycons atthe reducing end. Excluding these resonances, the averages of thedifferences in chemical shifts between the FCHASE glycosides and theirreference compound were less than ±0.2 ppm. Hence, comparison of protonchemical shifts, J_(HH) values and ¹³C chemical shifts with knownstructures, and use of NOEs or HMBC were all used to determine thelinkage specificity for various glycosyltransferases. The advantage ofusing HSQC spectra is that the proton assignment can be verifiedindependently to confirm the assignment of the ¹³C resonances of theatoms at the linkage site. In terms of sensitivity, the proton NOEs arethe most sensitive, followed by HSQC then HMBC. Using a nano-NMR probeinstead of a 5 mm NMR probe on the same amount of material reducedconsiderably the total acquisition time, making possible the acquisitionof an HMBC experiment overnight.

Discussion

In order to clone the LOS glycosyltransferases from C. jejuni, weemployed an activity screening strategy similar to that which wepreviously used to clone the α-2,3-sialyltransferase from Neisseriameningitidis (Gilbert et al., supra.). The activity screening strategyyielded two clones which encoded two versions of the sameα-2,3-sialyltransferase gene (cst-I). ORF analysis suggested that a 430residue polypeptide is responsible for the α-2,3-sialyltransferaseactivity. To identify other genes involved in LOS biosynthesis, wecompared a LOS biosynthesis locus in the complete genome sequence of C.jejuni NCTC 11168 to the corresponding locus from C. jejuni OH4384.Complete open reading frames were identified and analyzed. Several ofthe open reading frames were expressed individually in E. coli,including a β-1,4-N-acetylgalactosaminyl-transferase (cgtA), aβ-1,3-galactosyltransferase (cgtB) and a bifunctional sialyltransferase(cst-II).

The in vitro synthesis of fluorescent derivatives of nanomole amounts ofganglioside mimics and their NMR analysis confirm unequivocally thelinkage specificity of the four cloned glycosyltransferases. Based onthese data, we suggest that the pathway described in FIG. 4 is used byC. jejuni OH4384 to synthesize a GT1a mimic. This role for cgtA isfurther supported by the fact that C. jejuni OH4342, which carries aninactive version of this gene, does not have β-1,4-GalNAc in its LOSouter core (FIG. 1). The cst-II gene from C. jejuni OH4384 exhibitedboth α-2,3- and α-2,8-sialyltransferase in an in vitro assay whilecst-II from C. jejuni O:19 (serostrain) showed onlyα-2,3-sialyltransferase activity (Table 5). This is consistent with arole for cst-II in the addition of a terminal α-2,8-linked sialic acidin C. jejuni OH4382 and OH4384, both of which have identical cst-IIgenes, but not in C. jejuni O:19 (serostrain, see FIG. 1). There are 8amino acid differences between the Cst-II homologues from C. jejuni O:19(serostrain) and OH4382/84.

The bifunctionality of cst-II might have an impact on the outcome of theC. jejuni infection since it has been suggested that the expression ofthe terminal di-sialylated epitope might be involved in the developmentof neuropathic complications such as the Guillain-Barré syndrome(Salloway et al. (1996) Infect. Immun. 64, 2945–2949). It is also worthnoting that its bifunctional activity is novel among thesialyltransferases described so far. However, a bifunctionalglycosyltransferase activity has been described for the3-deoxy-D-manno-octulosonic acid transferase from E. coli (Belunis, C.J., and Raetz, C. R. (1992) J. Biol. Chem. 267, 9988–9997).

The mono/bi-functional activity of cst-II and theactivation/inactivation of cgtA seem to be two forms of phase variationmechanisms that allow C. jejuni to make different surface carbohydratesthat are presented to the host. In addition to those small genealterations that are found among the three O:19 strains (serostrain,OH4382 and OH4384), there are major genetic rearrangements when the lociare compared between C. jejuni OH4384 and NCTC 11168 (an O:2 strain).Except for the prB gene, the cst-I locus (including cysN and cysD) isfound only in C. jejuni OH4384. There are significant differences in theorganization of the LOS biosynthesis locus between strains OH4384 andNCTC 11168. Some of the genes are well conserved, some of them arepoorly conserved while others are unique to one or the other strain. Twogenes that are present as separate ORFs (#5a: cgtA and #10a: NeuA) inOH4384 are found as an in-frame fusion ORF in NCTC 11168 (ORF #5b/#10b).β-N-acetylgalactosaminyltransferase activity was detected in thisstrain, which suggests that at least the cgtA part of the fusion may beactive.

In summary, this Example describes the identification of several openreading frames that encode enzymes involved in the synthesis oflipooligosaccharides in Campylobacter.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. An isolated or recombinantly produced protein comprising apolypeptide having β1,3-galactosyltransferase activity, wherein thepolypeptide is at least 95% identical to SEQ ID NO:
 29. 2. The isolatedor recombinantly produced protein of claim 1, wherein the amino acidsequence is SEQ ID NO:29.
 3. The isolated or recombinantly producedprotein of claim 1, further comprising a tag for purification.
 4. Theisolated or recombinantly produced protein of claim 1, wherein theprotein is recombinantly produced and at least partially purified. 5.The isolated or recombinantly produced protein of claim 1, wherein theprotein is expressed by a heterologous host cell.
 6. The isolated orrecombinantly produced protein of claim 5, wherein the host cell is E.coli.